Dpto. Biología Molecular e Ingeniería Bioquímica

The bipolar disjunction in biogeography: case studies in the genus Carex (Cyperaceae) La disyunción bipolar en biogeografía: casos de estudio en el género Carex (Cyperaceae)

TESIS DOCTORAL Tamara Villaverde Hidalgo Sevilla, 2015

Dpto. Biología Molecular e Ingeniería Bioquímica

The bipolar disjunction in biogeography: case studies in the genus Carex (Cyperaceae) La disyunción bipolar en biogeografía: casos de estudio en el género Carex (Cyperaceae)

Memoria presentada por la licenciada en Biología Tamara Villaverde Hidalgo para optar al título de Doctora en Estudios Medioambientales (Doctorado Internacional) por la Universidad Pablo de Olavide de Sevilla. Sevilla, Julio 2015 Directores

Dr. Modesto Luceño Garcés

Dr. Santiago Martín Bravo Dr. Marcial Escudero Lirio

Agradecimientos

Para poder llegar hasta el volumen que tienes ahora en las manos, ha sido necesaria la ayuda de muchas personas. Éste ha sido un camino muy duro, aunque también muy enriquecedor, que no hubiera sido posible terminar sin las manos prestadas por los siguientes compañeros de viaje:

Mis directores de tesis. Modesto, muchas gracias por dejarme cumplir un sueño y darme la oportunidad de crecer profesionalmente. Hacer una tesis sin beca es una de las situaciones más indeseables para un doctorando. Gracias por creer que merecía la pena invertir en mí y por darme la posibilidad de estar en las aulas, ha sido una de las experiencias más gratificantes de mi vida. Siempre te estaré agradecida. Marcial y Santi, además de prestaros a ser mi brújula y mi mapa, habéis puesto el ímpetu y las ganas para llegar hasta aquí. Gracias por subiros al barco, cuidarme tanto y darme vuestro afecto. Ha sido un verdadero placer aprender a vuestro lado. Gracias de todo corazón.

Mis compañeros de laboratorio. Enrique, Inés, Jose, Marcial, Modesto, Mónica, Paco,

Pedro y Santi, sois mi familia en la UPO. Gracias por haber actuado como una válvula de escape al estrés de la tesis, a veces, volviéndome a poner los pies en la tierra, y otras, haciéndome reír hasta perder la respiración. He tenido mucha suerte llegando a un grupo como el vuestro, donde siempre he contado con vuestra ayuda y cariño. ¡Gracias! Sir

Henry, me siento muy afortunada por haberte conocido y por haber encontrado en ti un hombro (el de un gigante) en el que apoyarme durante este camino. Gracias por ser una persona tan maravillosa conmigo. ¡MagVilla’s! También quiero darle las gracias a esos compañeros que han pasado por el laboratorio y con los que he pasado uno buenos momentos: Carlos, Carmen, Cristina, Flo, Gloria, Laura,

Manu, Nacho, Paloma, Samuel, Víctor… y a todos los estudiantes del área de botánica que han estado conmigo estos años. Compañeros de otros laboratorios. Quiero darle las gracias a todo el equipo de Andrew

Hipp (The Morton Arboretum): Andrew, Bethany, Elisabeth, Marlene, grupo de voluntarios, Elisabeth Li (biblioteca) y demás compañeros, que han hecho que mi estancia en Chicago haya sido fabulosa. ¡Gracias por vuestra ayuda y por enseñarme tantas cosas!

Quiero darle también las gracias a las personas que han recolectado para estos trabajos

(Leo Bruederle, Pedro Jiménez Mejías, Mihai Pusças, Wayne Sawtel, Pablo Vargas,), y a todos los conservadores de los herbarios que nos han dado acceso a sus colecciones. También quiero darle las gracias a Paco Rodríguez Sánchez (Estación

Biológica de Doñana – CSIC) por su ayuda y comentarios a los análisis de nicho ecológico así como a José Luis Blanco Pastor por su ayuda con el programa Maxent.

Gracias a todos los investigadores del Canadian Museum of Nature, Jeff Saarelay

Lynn Gillespie, por ayudarme con los trabajos de morfometría y a Michel Gosselin por facilitarme mucha bibliografía ornitológica. También a mis compañeros de laboratorio: Anna, Jocelyn, Katia, Neda, Paul, Roger, Wayne...

Mis amigos. Tengo un grupo de cinco amigas de toda la vida a las que quiero agradecer que siempre hayan estado, y estén, ahí. Carmen, Campano, María, Martínez y Porti, gracias por ser mis Malvadas hermanas postizas. Llegar hasta aquí ha sido gracias a tardes llenas de risas y sabios consejos. Tengo otro grupo de amigos que, aunque hayan llegado un poco más tarde, son los culpables de ponerme un sonrisote y una cerveza en la mano estos años de tesis: Cobos, Coco, Vero, Edu, Eli, Elisa, Esteban, Estrella,

Jeovani, Maite, Raquel, Rosa, Paco, compis de la carrera, compis del máster en la UPO, compis de Toastmaster Sevilla, … Mi familia. Tengo la familia más molona del mundo mundial. Sois mi refugio, mi ejemplo de esfuerzo y mi inspiración diaria. Gracias por ser incondicionales y buscar los medios, las palabras y los abrazos necesarios para animarme a perseguir mis metas en la vida.

A mis padres.

A mis hermanos.

ÍNDICE

Abstract / Resumen …………………………………………………………… 1 Chapter 1. Introduction ……………………………………………………. 1 Biogeography …………………………………………………………….  Bipolar plant disjunctions ……………………………………………… 22 Hypothesis tested in bipolar disjunctions ……………………………… 23 Molecular markers for biogeographical studies and the need of divergence analyses ………………………………………………………………… 28 Carex (Cyperaceae), the genus with the greatest number of bipolar species ……………………………………………………………………. 30 Objetives by chapters …………………………………………………… 36 References ……………………………………………………………… 38 Appendix S1 ……………………………………………………………. 47

Chapter 2. Taxonomy of the Carex capitata complex………………………… 59 Abstract ………………………...……………………………………… 62 Introduction ………………………………………………………………63 Materials and methods …………………………………………..…… 64 Results …………………………………………………………….…… 68 Discussion ……………………………………………………………...... 75 References ……………………………………………………………….. 93 Appendix S1 ……………………………………………………………... 97 Additional Information ……………………………………………...…... 123

Chapter 3. Direct long-distance dispersal best explains the bipolar distribution of Carex arctogena (Carex sect. Capituligerae, Cyperaceae) ……………………. 155 Abstract ……………………………………………………………...... 154 Introduction ……………………………………………………….....…. 154 Materials and methods ………………………………………………159 Results ………………………………………………………………….... 160 Discussion …………………………………………………………...... 162 Acknowledgements ……………………………………………………. 166 References …………………………………………………………….... 166 Appendix S1 ………………………………………………………….….. 169

Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar disjunction of Carex maritima (Cyperaceae)……………………... 187 Abstract ………………………………………………………………... 189 Introduction ……………………………………………………………. 189 Materials and methods ………………………………………………… 190 Results …………………………………………………………………. 192 Discussion …………………………………………………………….. 195 Acknowledgements ……………………………………………………. 198 References ……………………………………………………………... 198 Appendix S1 …………………………………………………………... 201 Appendix S2 …………………………………………………………...... 217

Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread Carex bipolar species: biogeography of C. canescens Cyperaceae) …………………………………………………………………………………… 229 Abstract …………………………………………………………………... 232 Introduction ………………………………………………………………. 234 Materials and methods ………………………………………………… 236 Results …………………………………………………………………. 240 Discussion ……………………………………………………………...... 243 Acknowledgements ……………………………………………………. 247 References ……………………………………………………………... 248 Appendix S1 ……………………………………………………………... 261 Appendix S2 ……………………………………………………………... 275 Capítulo 6. Discussion and conclusions ……………………………………….. 283 Carex arctogena is a bipolar species …………………………………….. 285 Geological and climatic changes since the Miocene that allowed Northern and Southern…………………………………………………………………... 292 Direct long-distance dispersal vs. mountain-hopping …………………. 296 North to South long-distance dispersal ………………………………... 297 Means of dispersal ……………………………………………………….. 300 Successful establishment after dispersal in Carex bipolar species………. 305 Conclusions ………………………………………………………………. 308 References ………………………………………………………………... 310

Abstract

At a global level, one of the most fascinating plant distribution patterns is the bipolar disjunction. Bipolar species are defined here as species occurring at very high latitudes

(>55ºN and >52ºS) in both hemispheres, regardless of their distribution in intermediate areas. Under these criteria, around 30 vascular plant species have such distribution, being Carex (Cyperaceae) the genus with the largest number of bipolar species (six).

We performed a biogeographic study on three of them (C. arctogena, C. maritima and

C. canescens), based on morphological, molecular and bioclimatic data to shed light on the origin of their bipolar distribution. The four traditional hypotheses accounting for this pattern were tested: vicariance, direct long-distance dispersal, mountain hopping and convergence / parallel evolution. Methods used to accomplish this objective include molecular phylogenetic and phylogeographic analyses, divergence time estimation analyses, uni- and multivariate morphometric analyses, and species niche modelling.

The low levels of genetic differentiation found between populations of both Hemisphere and relatively recent times of diversification allow rejecting all but the long-distance dispersal hypothesis (including direct long distance dispersal and mountain hopping) for the studied Carex bipolar species. The studied species probably migrated from the

Northern Hemisphere to the Southern Hemisphere. In the case of C. canescens, two independent dispersal events were needed to achieve its current distribution.

Resumen

A nivel global, uno de los patrones de distribución más fascinantes corresponde a la disyunción bipolar. Las especies bipolares se definen en este trabajo como aquellas que se distribuyen a muy altas latitudes (>55ºN y >52ºS) en ambos hemisferios, independientemente de tener poblaciones a latitudes intermedias. Bajo estos criterios,

15 aproximadamente 30 especies de plantas vasculares presentan esta distribución, siendo el Carex (Cyperaceae) el género con mayor número de especies bipolares (seis). Hemos realizado un estudio biogeográfico en tres de ellas (C. arctogena, C. maritima y C. canescens), basándonos en datos morfológicos, moleculares y bioclimáticos para aportar evidencias sobre el origen de sus disyunciones bipolares. Testamos las cuatro hipótesis tradicionalmente propuestas para explicar este patrón: vicarianza, dispersión directa a larga distancia, saltos entre montañas, y evolución paralela o convergente. Los métodos usados para alcanzar este objetivo incluyen análisis moleculares filogéneticos y filogeográficos, análisis de estimación de tiempos de divergencia, análisis morfométricos uni- y multivariables, y modelización de nicho. Los bajos niveles de diferenciación genética encontrados entre las poblaciones de ambos hemisferios, así como los relativos recientes tiempos de diversificación de las especies estudiadas nos permiten rechazar todas las hipótesis excepto la dispersión a larga distancia (incluyendo dispersión directa y por salto de montañas). Las especies estudiadas probablemente migraron del Hemisferio Norte al Hemisferio Sur. En el caso de C. canescens, dos eventos de dispersión independientes fueron necesarios para alcanzar su distribución actual.

16 Chapter 1

Introduction

17 18 ______Chapter 1. Introduction

Introduction

Biogeography

Darwin (1809 – 1882; 1859, p.1) begins the Origin of the Species concerned about the information that could be gathered from the historical and geographical distribution of organisms in the light of his theory. Distribution was an important dimension of Darwin’s theory of evolution: “When on board of H.M.S. Beagle, as a naturalist, I was much struck with certain facts in the distribution of the inhabitants of South America, and in the geological relations of the present to the past inhabitants of that continent. These facts seemed to me to throw some light on the origin of species”. He did not only find dispersal as the most plausible explanation for the distribution of organisms [“ …the view of each species having produced in one area alone, and having subsequently migrated from that area as far as its power of migration and subsistence under past and present conditions permitted, is the most probable” (Darwin, 1859, p. 353)], but he also highlighted it as a key element shaping species range [“…all the grand leading factors of geographical distribution are explicable on the theory of migration (generally of the more dominant forms of life), together with subsequent modification and the multiplication of forms. We can then understand that high importance of barriers, whether of land or water, which separate our several zoological and botanical provinces” (Darwin, 1859, p. 409)]. He devoted two out of 15 chapters of the

Origin of the Species to the study of the distribution of taxa over geographic space and time, this is, to biogeography. Therefore, biogeography may be understood as a key element on

Darwin’s theory of evolution. He emphasized three points: (1) barriers to migration allowed time for the slow process of modification through natural selection; (2) the concept of single centres of creation was critical; that is, each species was first originated in a single area only,

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and from that centre it would extend as far as its colonization ability would permit; (3) dispersal was a phenomenon of overall importance.

Alexander von Humboldt (1769 – 1859), often recognized as the father of plant biogeography

(Brown and Lomolino, 1998), and many other illustrious researchers such as Alfred Wallace

(1823 – 1913) or, more recently, Robert MacArthur (1930 – 1972) and Edward Wilson (1929

–) were captivated by this discipline. It started as a descriptive science, mapping the major vegetation types and their associated fauna, then adding diversity patterns along different gradients (e.g. latitudinal or elevation) to finally become a multidisciplinary science that links fields such as systematics, ecology, paleontology or climatology (Morrone, 2009). It has allowed the designation of the biogeographic realms – those areas into which the Earth can be divided given the distinct characteristics of flora and fauna found in each area (Figure 1).

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◄ Figure 1. Biogeographical kingdoms and regions of the world from Morrone (2002). 1–2,

Holarctic kingdom (= Laurasia): 1, Nearctic region; 2, Palaearctic region; 3–6, Holotropical kingdom (= eastern Gondwana): 3, Neotropical region; 4, Afrotropical region; 5, Oriental region; 6, Australotropical region. 7–12: Austral kingdom (= western Gondwana): 7, Andean region; 8, Cape or Afrotemperate region; 9, Antarctic region; 10, Neoguinean region; 11,

Australotemperate region; 12, Neozelandic region.

Biogeography may be considered either a synthetic (Brown & Lomolino, 2011) or an interdisciplinary (Morrone, 2009, Figure 2) discipline, and for this reason, biogeography is regarded as heterogeneous in its principles and methods, lacking the conceptual unity of other sciences (Morrone, 2009). Biogeography is divided in two categories: ecological and historical biogeography (Sanmartín, 2012).

◄ Figure 2. Interdisciplinary

situation of biogeography, at

the intersection of 6 different

disciplines (modified from

Morrone, 2009).

Ecological biogeography is concerned with ecological processes occurring over short temporal and small spatial scales (Myers and Gillers, 1988). In contrast, when dealing with evolutionary processes that concerns large time scales (i.e. millions of years) and large or global geographic scales, we run into historical biogeography (Crisci, 2001). Historical biogeography attempts to reconstruct the origin of taxa, this is, it addresses the how, when,

21 ______Chapter 1. Introduction

and why of species distributions (Jablonski et al., 1985). It is concerned about taxa’s sequences of dispersal, isolation, and extinction; and to explain how geological events have shaped their present-day distribution (Myers & Giller, 1988). Important questions are why a taxon is absent from apparently suitable areas beyond its present range, and how taxa have become spatially separated or disjunct (Giller et al., 2004). It is hypothesized that such patterns can be naturally caused by the break-up of a once continuous range (vicariance), by long-distance dispersal, or through independent origins of the taxon in two or more places

(parallelism or convergence). If we level down to microevolution, we encounter phylogeography, which concentrates on the geographic distribution of genealogical lineages, especially those within and among closely related taxa (Avise, 2000). At this level, the coalescence theory help to model genealogies within populations; in population genetics, it is applied to several individuals sampled from one population whereas in phylogenetics, only one individual is often sampled per population (as individuals from the same population are usually assumed to be genetically similar compared to the differences that exist among populations or species, Degnan & Rosenberg, 2009).

Bipolar plant disjunctions

Disjunct distribution of species is defined as any discontinuous distribution in which some parts of the species (or taxa) range are clearly separated from another part (Morrone, 2009).

One of the most fascinating plant distribution patterns concerning the Southern Hemisphere encompasses the bipolar disjunction. Bipolar species are defined in this work as species growing at very high latitudes (>55ºN and >52ºS) in both hemispheres, regardless of their distribution in intermediate areas (Moore & Chater, 1971). Under these criteria, there are only around 30 vascular plant species from 12 families that could be considered bipolar (Appendix

22 ______Chapter 1. Introduction

S1). Species circumscription in some of these cases are still poorly understood, therefore, some of them might leave this list after a taxonomic revision, whereas some other might join it after we gain a broader taxonomic knowledge on the Floras of both hemispheres. From the compilation of bipolar species by Moore & Chater (1971), there are currently at least seven species whose bipolar distribution is suspected to have an anthropogenic origin in one of the

Hemispheres (Appendix S1).

The families with the largest number of bipolar species are Poaceae Barnhart (8 species,

26.7%) and Cyperaceae Juss. (6 species, 20%); and the genus with the greatest number of bipolar species is Carex L. (6 species, 20%; Appendix S1). The majority of the bipolar species lacks molecular studies comparing Northern and Southern Hemisphere populations.

With the exception of the Carex bipolar species, none of the molecular studies concerning the remaining species have addressed specifically their bipolar distribution. The following studies have included at least one population from both hemispheres: Hymenophyllum tunbrigense (L.) Sm. (Hennequin et al., 2010), Anemone multifida Poir (Ehrendorfer et al.,

2009; Mlinarec et al., 2012), Triglochin palustre L. (von Mering, 2013), Avenella flexuosa

(L.) Dejer (it might be an introduction in South America; Chiapella, 2007) and Phleum alpinum L. (Boudko, 2014; see Appendix S1 for more details). Thus, these works could serve as a background to conduct more specific studies addressing the various hypotheses tested in bipolar distribution.

Hypothesis tested in bipolar disjunctions

Four hypotheses have historically been put forward to account for bipolar disjunctions: (1) stepwise long-distance dispersal across the equator and via mountain ranges (‘mountain- hopping’; (Raven, 1963; Moore & Chater, 1971; Ball, 1990; Heide, 2002; Vollan et al.,

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2006); (2) direct long-distance seed dispersal by birds, wind and/or ocean currents (Cruden,

1966; Muñoz et al., 2004; Nathan et al., 2008); (3) vicariance (Du Rietz, 1940), which implies a continuous distribution fragmentation dating back to the trans-tropical highland bridges during the Mesozoic Era (from the early Jurassic, 195 Ma; Scotese et al., 1988); and lastly, (4) convergent or parallel evolution of the disjunct populations (Scotland, 2011).

Long-distance dispersal

Darwin was so convinced by the hypothesis of migration over long-distances to account for species distributions, that he undertook a series of experiments to prove it (Darwin, 1859).

Since Darwin, the effectiveness of long-distance methods of seed dispersal has been documented broadly (e.g. Murray, 1986; De Queiroz, 2005; Nogales et al., 2012; Vargas et al., 2012). Seed dispersion patterns near sources can be qualitatively different from those far from sources, because dispersal processes can operate over different ranges of distances

(Nathan & Muller-Landau, 2000). Seed density around a mother plant almost invariably declines leptokurtically with distance (being more concentrated about the mean than the corresponding normal distribution), with an extended tail of long-distance dispersal (Harper,

1977; Willson, 1993; Nathan & Muller-Landau, 2000). The limited distances that most seeds travel are well documented for plants of all growth forms (e.g. Harper, 1977; Howe &

Smallwood, 1982; Willson, 1993a; Cain et al., 1998). Empirical data is mostly acquired for short-distance events than for rare long-distance dispersal events, due to the difficulty of sampling the latter (Nathan and Muller-Landau, 2000). However, short-distance dispersals cannot explain some observed patterns of genetic structure (Cain et al., 2000) or range expansion rates (Clark, 1998) and therefore, long-distance dispersal deserve its own sampling effort.

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Typically, several dispersal agents are involved in long-distance dispersal (Gillespie et al.,

2012), as Darwin suggested. Therefore, their seed shadows - the spatial distribution of seeds dispersed from a single plant (Nathan and Muller Landau, 2000)- are determined by the combined effects of displacement by all dispersal agents that move seeds from the parent plant (primary dispersal) or from subsequent locations (secondary dispersal). Wind current characteristics can be used to explain wind dispersal (e.g. distribution of plants in the

Southern Hemisphere might be affected by the West Wind Drift, Sanmartín et al., 2007, and references therein); however, animal behaviour, which could depend upon many variables

(e.g. abundances and characteristics of alternate food sources, competing species and predators), is typically more complex and it limits the understanding of zoochory (Nathan and

Muller-Landau, 2000). For instance, among the birds that void or defecate viable seeds, the attributes that most influence seed dispersal are behavioural rather than morphological or physiological (e.g. Howe & Estabrook, 1977; Herrera, 1984a, 1984b). Some authors (Ouborg et al., 1999; Cain et al., 2000) have emphasized the potential of genetic methods that can provide evidence of long-distance gene flow, either by comparing the genotypes of seedlings with potential parents or by examining genetic structure within and among populations

(Ouborg et al., 1999; Jordano & Godoy, 2000). Le Corre et al. (1997) showed that long- distance dispersal events influence the genetic differentiation of populations, leaving a genetic signature that could persist for long periods of time. Moreover, it has been recently showed that DNA barcoding can help to identify the source plant of the dispersed seeds and the frugivore species that contribute to each dispersal event (González-Varo et al., 2014), which has an extraordinary potential for characterizing long-distance dispersal in plants.

25 ______Chapter 1. Introduction

Direct vs. mountain-hopping long-distance dispersal

The main difference between direct and mountain-hopping long distance dispersal of plants is the number of “way stations” made before reaching the end of the dispersal process. In the bipolar disjunction, direct long-distance dispersal implies that the taxa have been carried from its source in one side of the disjunction to the other, without any stop between these areas.

The mountain-hopping hypothesis (Ball, 1990) proposes a long-distance, stepwise migration of these taxa using mountains peaks as stepping-stones to cross the tropics. Means of dispersal in direct long-distance dispersal events could be the same as in mountain-hopping ones.

Vicariance

Vicariance is defined as the splitting of the continuous geographical range of a group into two or more parts by the development of some sort of barrier (or barriers) to dispersal (de

Queiroz, 2005). The fossil record can be used to evaluate vicariance and dispersal hypotheses by dating lineage divergences (nodes). In dated phylogenetic reconstructions, we encounter two categories of results: (1) a particular evolutionary branching point is estimated to be as old as or older than the fragmentation event in question, that node is supporting a vicariant event; (2) a branching point is estimated to be younger than the fragmentation event, then, it is supporting long-distance dispersal. The biogeographic history of the Southern Hemisphere is considered a prime example of the vicariance scenario (Sanmartín & Ronquist, 2004). The disjunct trans-Pacific distributions have been proposed to stem from the sequential breakup of the southern supercontinent Gondwana during the last 165 million years. This hypothesis has been long tested in angiosperm groups with Southern Hemisphere distributions (reviewed in

Beaulieu et al., 2013) and it has been supported in some plant groups [e.g. in the genus

26 ______Chapter 1. Introduction

Gunnera (Gunneraceae), Wanntorp & Wanntorp, 2003; in the family Myrtaceae, Sytsma et al., 2004]. Although dispersal and vicariance are often considered competing hypotheses in biogeography (Sanmartín and Ronquist, 2004), both are usually claimed to explain Southern

Hemisphere plant distribution (e.g. Nothofagus, Knapp et al., 2005). For bipolar species, we consider the fragmentation of a continuous distribution dating back to the trans-tropical highland bridges during the Mesozoic Era (from the early Jurassic, 195-200 Ma; Scotese et al., 1988; Figure 3). During this time, bipolar species could have had a continuous distribution from high latitudes in the Northern Hemisphere to high latitudes in the Southern

Hemisphere.

Figure 3. Landmasses in the early Jurassic (ca. 200 Ma; photo taken from Scotese (2004).

After an episode of igneous activity along the east coast of North America and the northwest coast of Africa, the central Atlantic Ocean opened as North America moved to the northwest.

This movement also gave rise to the Gulf of Mexico as North America moved away from

South America (Scotese, 2002).

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Convergent and parallel evolution

Traditionally, ‘convergent’ has been distinguished from ‘parallel’ evolution as the first assumes that when a given phenotype evolves, the underlying genetic mechanisms are different in distantly related species (convergent evolution; Haldane, 1932) but similar in closely related species (parallel evolution; Haldane 1932). There is still a huge debate between parallel evolution and convergence (e.g. (Wichman et al., 1999; Cooper et al., 2003;

Fong et al., 2005; Christin et al., 2007; Scotland, 2011). If we assume that homoplasy can be seen as convergence in a broad sense, then pheonotypic homoplasy can be described as convergence and genotypic homoplasy as parallelism (Scotland, 2011). However, Stern

(2013; and references therein) showed, on one hand, several examples where the same phenotype (e.g. coloration in lizards) might evolve among populations within a species by changes in different genes; on the other hand, he showed examples of similar phenotypes that might have evolved in distantly related species by changes in the same gene. Therefore, it is argued that ‘convergent’ and ‘parallel’ evolution represents ends of a continuum and both can be described with a single term – convergent evolution (Stern, 2013). For bipolar taxa, we will consider convergent or parallel evolution as synonym hypotheses that can be rejected is taxa are retrieved as monophyletic.

Molecular markers for biogeographical studies and the need of divergence analyses

When conducting studies in biogeography, ordered markers (DNA sequences) are preferred rather than unordered ones (e.g. AFLP, ISSR, RAPD) because the first contain records of their own histories and provide information about genealogical relationships (e.g. Schaal &

Olsen, 2000; Schaal & Leverich, 2001).

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The chloroplast genome has a lower degree of polymorphism than the nuclear genome

(Muse, 2000), however, it can uncover a higher degree of genetic structure. In the chloroplast genome, genetic drift occurs more rapidly than in the nuclear because it has a lower effective population size (Louis et al., 1998); thus, genetic drift result in greater genetic differentiation of fragmented populations, retaining molecular signal of past migrations, dispersal events and range fragmentation (e.g. Louis et al., 1998; Newton et al., 1999; Hudson & Coyne, 2002;

Rendell & Ennos, 2003; Kadereit et al., 2005; Petit et al., 2005). This difference is due to the uniparental inheritance of chloroplast DNA (cpDNA), which is maternally inherited in most angiosperms (Harris & Ingram, 1991). In plant biogeographical studies, as colonization of new habitats commonly occurs through seeds, the pattern of dispersal is unaffected by subsequent pollen movements and may be traced with cpDNA markers (Petit et al., 2003).

Moreover, the chloroplast genome is represented by only one DNA molecule where recombination processes are scarce. Conversely, nuclear markers may contain multiple different regions of the nuclear genome with more frequent recombination events between those regions. Therefore, nuclear markers are generally more useful for exploring the recent history of taxa and gene flow patterns of species (Harpending et al., 1998).

Finally, next-generation sequencing techniques (e.g. restriction-site associated DNA; Baird et al., 2008) are now being used in biogeographic studies (e.g. Emerson et al., 2010; Lexer et al., 2013).

Divergence time analysis and the use of local molecular clocks is now a basic tool in biogeography (Givnish & Renner, 2004). It has allowed to test different hypothesis in plant disjunctions (e.g. Winkworth et al., 2002; Sytsma et al., 2004; Wen & Ickert-Bond, 2009; Nie et al., 2012) and discern between dispersal or vicariance, the two main hypotheses tested in biogeography, in many different groups of angiosperms (e.g. Malpighiaceae, Davis et al.,

2002; Moraceae, Zerega et al., 2005; Ephedra, Ickert-Bond et al., 2009). These studies are

29 ______Chapter 1. Introduction

typically supported by fossil records which are ideal to estimate the ages of lineages (acting as calibration points) and to support their presence in particular places (Renner, 2005).

However, there are many plant groups lacking reliable fossil records and, therefore, secondary calibrations are broadly used (e.g. (Valente et al., 2011; Fernández-Mendoza &

Printzen, 2013; Pimentel et al., 2013; Inda et al., 2014). In the case of bipolar species, divergence time estimates can help to elucidate between the different hypotheses tested.

Carex (Cyperaceae), the genus with the greatest number of bipolar species

One of the families with the largest number of bipolar species is the Cyperaceae. The sedge family is also among the largest families of flowering plants, occurring on all continents, except Antarctica. It comprises approximately 104 genera, 14 tribes and 5,400 species

(Goetghebeur, 1996) making it the 7th or 8th largest angiosperm family and the third largest monocot family after orchids (Orchidaceae Juss.) and grasses (Poaceae). Its species occur in a great diversity of habitats, ranging from deserts to rainforests (Reznicek, 2011), although they are predominantly found in wetland habitats such as littoral communities, peat-lands and wet meadows. Although its economic significance is often at a regional or local level (Simpson &

Inglis, 2001), approximately 10% of its species are used by humanity for food (Chinese water chestnut, Eleocharis dulcis (Burm.) f. Trin. ex Henschel, or the yellow nut sedge, Cyperus esculentus L.); for pasture (Carex lyngbyei Hornem.); for construction (Schoenoplectus californicus (C.A. Mey) Palla); as an elixir (Carex arenaria L.); and even for making paper

(Cyperus papyrus L.), whereas other sedge species, such as Cyperus rotundus L., C. esculentus L., C. difformis or Fimbristylis miliacea L. are considered to be serious weeds due to their negative effect on agriculture (Brayson & Carter, 2008).

30 ______Chapter 1. Introduction

Sedge flowers are highly small and evolutionary reduced in size typically with the perianth lacking or reduced to either bristles or scales. In Carex, a modified bract in the female flower surrounds the naked gynoecium, enclosing the pistil and later the achene, in a sac-like structure (Blaser, 1944); known as utricle or perigynium. The flowers are arranged in structures known as spikelets with the inflorescence consisting of one or many spikelets arranged on one or more axes. Approximately 40% of all sedge species (ca. 2100 spp.) are grouped in the cosmopolitan tribe Cariceae Kunth ex Dumort., which has been suggested by most studies to be sister to tribe Scirpeae or nested within it (e.g. Muasya & Simpson, 1998;

Muasya et al., 2009; Escudero & Hipp, 2013; Hinchliff & Roalson, 2013; Jung & Choi, 2013;

Léveillé-bourret et al., 2014). (Waterway and Starr (2007), using DNA from both nuclear and plastid genomes, revealed three major clades within Cariceae that roughly corresponded to:

(1) subgenus Vignea, hence named Vignea clade; (2) subgenera Carex and Vigneastra, named the Core Carex clade; and (3) most unispicate Carex species plus Cymophyllus,

Kobresia, Schoenoxiphium, and Uncinia, named the Caricoid clade (Figure 3). Later on,

Waterway et al., (2009) found that section Siderostictae Franch. ex Ohwi, traditionally classified in subgenus Carex, formed, together with the Hypolytroides clade (Starr et al.,

2015), a clade sister to all other species in tribe Cariceae; it confirmed that Carex was a paraphyletic group with all other genera of tribe Cariceae nested within it. For these reasons, it has been recently agreed by the (Global Carex Group, 2015) to consider a new broader circumscription of Carex, changing its classification by unifying all genera within it.

31 ______Chapter 1. Introduction

Figure 4. Generalized phylogenetic tree of Cyperaceae tribe Cariceae based on molecular phylogenetic studies to date (modified from Global Carex Group, 2015). Solid lines show relationships that are supported by all or most studies; dotted branches show relationships that are frequently seen but more inconsistent among studies; branches with consistently high boostrap support are indicated with a grey filled circle.

Therefore, the cosmopolitan genus Carex, the most diverse angiosperm genus of the northern temperate zone (Escudero et al., 2012b) is the largest genus in the family and it is also one of the most taxonomically difficult (Starr & Ford, 2009) due to its complex and extremely reduced morphology. Different potential drivers of diversification have been proposed to contribute to the extraordinary diversity of Carex [e.g. self-compatibility and high selfing

32 ______Chapter 1. Introduction

rates (Arens et al., 2005; Friedman & Barrett, 2009; Escudero et al., 2010b) or chromosome differentiation (Whitkus, 1988; Hipp, 2007; Escudero et al., 2010b, 2012a, 2012b, 2013a,

2013b; Hipp et al., 2010; Jiménez-Mejías et al., 2012)].

A comprehensive, global taxonomic treatment of the genus is still lacking, and new species continue to be described. Thus, in the last 20 years, the discovery rate of new Carex species in North America has been, on average, two per year (Starr & Ford, 2008), which seems to be a trend that has not yet reached a plateau.

There are six bipolar Carex species (Figure 5): Carex arctogena Harry Sm., C. canescens L.,

C. macloviana D’Urv., C. magellanica Lam., C. maritima Gunn. and C. microglochin

Wahlenb. These species are placed in different lineages within the genus. In the clade

Caricoid, there is C. arctogena and C. microglochin; in the Vignea clade, C. canescens, C. macloviana, C. maritima; and in the core Carex clade, C. magellanica. Therefore, this extraordinary geographic disjunction seems to have been achieved independently by Carex species from different evolutionary lineages. None of this species but C. microglochin present specialized dispersal devices (a ‘hook’ used for ectozoochory; Savile, 1972); however, it has been proved that, for instance, epizoic dispersal occurs in other Carex species without having evident morphological features for it (reviewed in Allessio Leck & Schütz, 2005).

Carex bipolar species generally have a circumboreal distribution and are limited to austral latitudes in South America (>52º; Figure 5). An exception is C. canescens (sect. Glareosae

G. Don), the single bipolar Carex species that reaches not only the southernmost region of

South America (Tierra del Fuego and Falkland Islands) but also Oceania (including Australia,

Tasmania and New Guinea; Figure 1 and Appendix S1), occurring within five biogeographical regions (Nearctic, Palearctic, Andean, Neoguinean and Australotemperate;

Morrone, 2002). Carex canescens is therefore the bipolar Carex species with the widest

33 ______Chapter 1. Introduction

distribution followed by C. maritima, which has a circumboreal distribution including the

European Alps and the Himalayas in the Northern Hemisphere, while in the Southern

Hemisphere it is distributed from Ecuador to Patagonia (Govaerts et al., 2014).

All species but C. arctogena were studied molecularly by Vollan et al. (2006) and Escudero et al. (2010a), although with a limited sampling. Both studies found low levels of genetic differentiation between populations from different Hemispheres, suggesting that either mountain-hopping or direct long-distance dispersal was the best explanation for the species’ current distributions. However, neither Vollan et al. (2006) nor Escudero et al. (2010) could determine definitively which hypothesis best explained the distributions of bipolar species.

34 ______Chapter 1. Introduction

Figure 5. Distribution maps of the six bipolar Carex species. (a) Carex arctogena; (b) C.

canescens; (c) C. macloviana; (d) C. magellanica; (e) C. marítima; and (f) C. microglochin.

The dark grey regions indicate the distribution obtained from the World Checklist of Selected

Plant Families (http://apps.kew.org/wcsp).

35 ______Chapter 1. Introduction

Objectives by chapters

1. The main goal of Chapter 2 was to resolve taxonomic problems within the C. capitata

complex, especially in relation to the status of the different taxa described within this

complex. Morphological, micromorphological, ecological and geographical data are

studied using more than 450 herbarium specimens.

2. The goal of Chapter 3 was to determine which of the four classic hypotheses used to

account for bipolar taxa could best explain the distribution of C. arctogena. By

evaluating the combined evidence provided by phylogenetic reconstructions and

molecular dating based on nuclear and plastid data together with bioclimatic data

through species’ distribution, biogeographical hypotheses were tested, improving our

understanding of the historical events that promoted the formation of the bipolar

disjunction seen in C. arctogena.

3. The aim of Chapter 4 was to explain the bipolar distribution of C. maritima.

Specifically, the aims were: (i) to clarify the direction of the dispersal (north-to-south

or south-to-north); (ii) in the case of genetic structure, to estimate the timing of

dispersal; and (iii) to test mountain-hopping and direct long-dispersal hypotheses, as

well as the relationship of C. maritima with biotic and abiotic factors that could

explain the bipolar distribution. In order to accomplish this task data on nuclear and

plastid molecular markers and bioclimatic data were combined. Carex maritima

populations were analysed phylogenetic, phylogeographically and ecologically

through its distribution.

36 ______Chapter 1. Introduction

4. The aims of Chapter 5 were to: (i) test the various hypotheses accounting for the

bipolar disjunction of C. canescens; and (ii) to determine whether C. canescens

migrated twice to the Southern Hemisphere or was dispersed from South America to

Australia or vice versa. Phylogenetic reconstructions and phylogeographical analyses

(based on nuclear and plastid regions) as well as bioclimatic data were evaluated in

the total distribution of the species.

5. In Chapter 6, the objectives were to: (i) to review the hypotheses tested in bipolar

distribution; (ii) to infer the most common direction of dispersal in bipolar species;

(iii) to discuss about the possible means of dispersal that could have promoted bipolar

distribution; and (iv) to highlight the possible characteristics of the bipolar species

that have made them successful in establishment after dispersal.

37 ______Chapter 1. Introduction

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46 ______Chapter 1. Introduction

Appendix S1

47 ______Chapter 1. ______Introduction

Appendix S1. Bipolar species list from Moore & Chater (1971). Monocot species distributions have been obtained from Goaverts et al. (2014; http://apps.kew.org/wcsp/ 2015-05-20.); otherwise, noted.

Family Species Distribution Status

Lycopodiaceae Huperzia selago (L.) Northern Hemisphere (North America and There are no molecular works comparing Bernh. ex Schrank & Eurasia), Macaronesia, Australia, Tasmania and populations from both hemispheres. A Mart. New Zealand (Villar, 1968). worldwide taxonomic study is needed due to its contrasting treatment in different floras (Aiken et al., 2007)

Hymenophyllaceae Hymenophyllum Western and Southern Europe, Macaronesia Although there is uncertainty about the tunbrigense (L.) Sm. (Sánchez-Velázquez, 2003), Africa (Gabon, circumscription of its populations in Asia, Kenya, Malawi, Tanzania, Mozambique, South Mexico, Central and South America (Richards & Africa, Swaziland, Zimbabwe, Madagascar and Evans, 1972; Farrar, 1993), Hennequin et al. Mauritius; Roux, 2001), New Zealand, Central (2010) showed that Chilean and Tanzanian and South America, Jamaica and in a single populations differed genetically from the locality in North America (South Carolina; European ones (Asian or Central American Farrar, 1993). populations were not included).

Hymenophyllum Widespread in the temperate regions of both Larsen et al. (2013) showed genetic differences peltatum (Poir.) hemispheres (Hnatiuk, 1972; Marticorena et al., only between Southern hemisphere populations. Desv. 2001; Roux, 2001), Africa (Kenia, South Therefore, a worldwide study is needed. Africa, Tanzania, Uganda and Marion and Prince Edward Islands), Australia, South America and Europe.

Polygonaceae Koenigia islandica North America and southern South America There are no molecular works comparing L. (Argentina, Chile); northern Europe; Central populations from both hemispheres.

48 ______Chapter 1. ______Introduction

and East Asia (Packer & Freeman, 2005).

Polygonum Europe, North of Africa, Southwest Asia, There are no molecular works comparing maritimum L. Macaronesia, South Africa and South America populations from both hemispheres. (Styles, 1962; Zuloaga et al., 2008).

Caryophyllaceae Cerastium arvense Temperate regions of North and South America There are no molecular works comparing L. (Moore 1983; Pedersen 1984; Wagstaff and populations from both hemispheres. It is a Taylor 1988; Hoffmann et al. 1997). In southern morphological complex taxon [treated as several South America, it has been described as exotic subspecies in Europe (Tutin et al. 1993); several and introduced (Brion et al. 1988; Volponi forms associated with differences in geographic 1990, 1999). distribution in North America (Hitchcock et al. 1977); and it has also been considered as extremely polymorphic in southern South America (Moore 1983; Pedersen 1984; Volponi 1990, 1999)]. Although Quiroga et al. (2002) showed morphological and genetic differences in the southern Andes populations and suggested ecotypic variation due to climatic changes during the Pleistocene, there are no molecular works supporting this assumption between hemispheres. Therefore, a worldwide study is needed.

Honckenya peploides Coastal North America and Eurasia. Its There are no molecular works comparing (L.) Ehrh. distribution in the Southern Hemisphere might populations from both hemispheres. be the result of anthropochorus origin (Sánchez- Vilas, 2007).

49 ______Chapter 1. ______Introduction

Sagina procumbens Originally from Europe, it has been introduced There are no molecular works comparing L. in North America (including Mexico), Central populations from both hemispheres. America (Costa Rica, Guatemala) and South America (Bolivia and southern Argentina) as well as in western Asia (Siberia) and Antarctica (sub-Antarctic Islands; Crow, 2005). It has invaded 14 out of 22 southern Oceanic Islands (Shaw et al., 2011) and its eradication in some of them is been considered (Cooper et al., 2011).

Ranunculaceae Ranunculus aquatilis Native in the Holarctic; introduced in the There are no molecular works comparing L. Southern Hemisphere (Cook, 1963; populations from both hemispheres. Whittemore, 1997; Ruiz, 2001; Eichler & Jeanes, 2007; Lumbreras et al., 2011).

Anemone multifida North and southern South America. There are great morphological differences across Poir. its distribution (Hoot et al., 1994). The genetic differences found between northern and southern hemispheres (Enhendorfer et al., 2009) might be the result of an ancestral hybridization and subsequent polyploidization (Meyer et al., 2010). It has been hypothesized a migration from North America to South America during the Quaternary (Mlinarec et al., 2012). In the northern hemisphere, allopolyploids were detected (Hoot et al., 2012; Mlinarec et al., 2012) as well as a high genetic variability between alpine vs. lowland ecotypes (McEwen

50 ______Chapter 1. ______Introduction

et al, 2013).

Plantaginaceae Plantago maritima North Africa, temperate Asia, North America There are no molecular works comparing L. and southern South America (Moore, Williams populations from both hemispheres. and Yates, 1972)

Hippuris vulgaris L. North America, southern South America and Although Chen et al. (2013) showed genetic Australia (Elven et al., 2012). variation between populations in Qinghai- Tibetan Plateau (China; Chen et al., 2013), there are no molecular works comparing populations from both hemispheres.

Plumbaginaceae Armeria maritima Predominantly Holarctic and southern South Although it has been widely studied genetically, (Mill.) Willd. America (Patagonia) ctyogenetically and molecularly (e.g. Weidema et al., 1996; Coulaud et al., 1999; Fuertes Aguilar & Nieto Feliner, 2003; Piñeiro et al., 2011; Abratowska et al., 2012), there are no molecular works comparing populations from both hemispheres.

Gentianaceae Gentiana prostrata North America, South America and Eurasia There are no molecular works comparing

51 ______Chapter 1. ______Introduction

Haenke populations from both hemispheres.

Scrophulariaceae Limosella australis Australia, North and South America; possibly There are no molecular works comparing R. Br. introduced in Europe (Wales; Jones, 2011) populations from both hemispheres.

Juncaginaceae Triglochin palustre Temperate areas of Eurasia, North America, Molecular studies based on nuclear (ITS) and L. South America and New Zealand. chloroplast regions (rbcL and matk) revealed no differentiation between northern and southern hemisphere populations and its distribution is suggested to be the result of a recent dispersal (von Mering, 2013).

Poaceae Catabrosa aquatica Temperate areas of Eurasia, North and South There are no molecular works comparing (L.) P. Beauv. America populations from both hemispheres.

Trisetum spicatum Australia, New Zealand, Eurasia, North and There are no molecular works comparing (L.) Richt. South America populations from both hemispheres.

Poa glauca Vahl. Eurasia and North America; introduced in South There are no molecular works comparing America (Giussani et al., 2012) populations from both hemispheres.

Vahlodea Eurasia, North and South America There are no molecular works comparing atropurpurea populations from both hemispheres. (Wahlenb.) Fr. ex Hartm. [=Deschampsia atropurpurea (Wahlenb.) Scheele]

52 ______Chapter 1. ______Introduction

Deschampsia Subarctic, temperate and tropical mountains of Chiapella (2007) shows some degree of cespitosa (L.) P. Eurasia, Africa, (but introduced in Cape molecular differentiation between northern and Beauv. Provinces and Lesotho); Australia (introduced southern hemispheres populations. in South Australia), North America (introduced in Hawaii) and South America [introduced in Bolivia, South Brazil, West and South Argentina, central and south Chile, Macquaire Islands and South Georgia (UK)]

Avenella flexuosa Eurasia, Africa (but introduced in Cape Chiapella (2007) shows some degree of (L.) Drejer Provinces) North America (introduced in molecular differentiation between northern and [=Deschampsia Hawaii, Aleutian Islands, Alaska, Yukon, southern hemispheres populations. flexuosa (L.) Trin.] British Columbia, Idaho, Oregon, Washington, Wyoming, Rhode Island, California, central Mexico), Central America (introduced in Costa Rica) and South America [introduced in central and South Brazil, Trista da Acunha (UK) and South Georgia (UK)]. Also introduced in North and South New Zealand.

Calamagrostis Temperate and subarctic Northern Hemisphere There are no molecular works comparing stricta (Timm.) Koel. (North America and Eurasia) and South populations from both hemispheres. America Bolivia, Peru and Patagonia)

Phleum alpinum L. Subarctic and temperate regions of the Northern Boudko (2014) showed no genetic differences Hemisphere (Eurasia and North America), between northern and southern hemisphere South America (Guatemala, Argentina and populations. Chile) and South Georgia

53 ______Chapter 1. ______Introduction

Cyperaceae Carex arctogena L. North America, Europe and southern South There are no molecular works comparing America (Patagonia) populations from both hemispheres.

C. canescens L. North America, Europe, Australasia and Vollan et al. (2006) and Escudero et al. (2010) southern South America (Patagonia) compared Northern and Southern Hemisphere and concluded that a long-distance dispersal as the best hypothesis to explain its bipolar distribution.

C. macloviana Eurasia, North America (including Hawaii) and Escudero et al. (2010) showed no molecular D’Urv. South America (Bolivia, Peru, Argentina and differences between Northern and Southern Chile) Hemisphere populations and concluded that a long-distance dispersal as the hypothesis that best explains its distribution.

C. magellanica Lam. Eurasia, North and South America (Patagonia) Escudero et al. (2010) showed molecular differences between Northern and Southern hemisphere populations and suggested that it has obtained its distribution by long-distance dispersal.

C. martima Gunn. North America, Eurasia and South America Escudero et al. (2010) compared Northern and Southern Hemisphere populations and suggested a long-distance dispersal as the most plausible hypothesis explaining its distribution.

C. microglochin Eurasia, North and South America (Colombia, Escudero et al. (2010) showed no molecular Wahlenb. Ecuador, Peru, Argentina and Chile) differences between Northern and Southern Hemisphere populations.

54 ______Chapter 1. Introduction

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57 ______Chapter 1. Introduction

58 Chapter 2

Taxonomy of the Carex capitata complex

59 60 ______Chapter 2. Taxonomy of the Carex capitata complex

Taxonomy of the Carex capitata complex

Tamara Villaverde1, Santiago Martín-Bravo1 and Julian R. Starr2,3

1Botany area, Department of Molecular Biology and Biochemical Engineering, Pablo de

Olavide University, ctra. de Utrera km 1, 41013, Seville, Spain, 2Canadian Museum of

Nature, PO Box 3443, Ottawa, ON K1P 6P4, Canada, 3Department of Biology,

Gendron Hall, University of Ottawa, 30 Marie Curie, Ottawa, ON K1N 6N5, Canada.

*Correspondence: Tamara Villaverde, Botany area, Department of Molecular Biology

and Biochemical Engineering, Pablo de Olavide University, ctra. de Utrera km 1,

41013, Seville, Spain.

E-mail: [email protected]

61 ______Chapter 2. Taxonomy of the Carex capitata complex

Abstract

Carex section Capituligerae is a small group placed in the Unispicate clade within the

Caricoid clade that comprises three to four taxa: Carex capitata L., an arctic-alpine

species with a circumpolar distribution; C. oreophila C. A. Mey, an alpine species

found in the mountains of western Asia and C. arctogena Harry Sm., an alpine species

from North America, South America and Eurasia. The taxonomy of the section has

traditionally been controversial, especially concerning the circumscription of the C.

capitata - C. arctogena species group. We performed uni- and multivariate analysis of

macro- and micromorphological characters (28 variables) from 450 specimens of the C.

capitata - C. arctogena species group, covering their morphological and geographical

variability, in order to elucidate its taxonomy. Carex capitata and C. arcotgena are

found to be morphologically different and populations from South America correspond

to C. arctogena. Morphological variability, which also corresponds with geographical

distribution, was found within populations from western North America and we suggest

the description of one species and two subspecies: Carex cayouetteana, C. cayouetteana

subsp. bajasierra and C. cayouetteana subsp. altasierra.

Keywords: arctic-alpine species, Carex arctogena, Carex capitata, Carex section

Capituligerae, species complex, morphology, PCA, taxonomy

62 ______Chapter 2. Taxonomy of the Carex capitata complex

Introduction

Recent molecular studies based on nuclear and plastid DNA sequences (ETS-1f, ITS,

trnL intron, trnL-trnF intergenic spacer) showed that there are four main clades within

the genus Carex (Waterway et al., 2009): (i) Vignea clade, which encloses all species in

the subgenus Vignea; (ii) Core Carex clade, which comprises subgenera Carex and

Vigneastra; (iii) Caricoid clade, which groups subgenus Psyllophora plus genera

Cymophyllus, Kobresia, Schoenoxiphium, and Uncinia; (iv) Siderostictae clade, formed

only by species in section Siderostictae. Later on, Starr et al., (2015) found another

clade, the Hypolytroides clade, sister to the Siderosticatae one, and both sister to all

other species in tribe Cariceae. All these results confirmed that Carex was a

paraphyletic group with all other genera of tribe Cariceae nested within it. For these

reasons, it has been recently agreed by the Global Carex Group (2015) to consider a

new broader circumscription of Carex, changing its classification by unifying all genera

within it. Therfore, the genera Cymophyllus, Kobresia, Schoenoxiphium, and Uncinia

were transferred into Carex. However, clade names are still used for Carex systematics.

Section Capituligerae is a small group placed in the Unispicate clade within the

Caricoid clade (Figure 4 in Chapter 1) that comprises three to four taxa: Carex capitata

L., an arctic-alpine species with a circumpolar distribution; C. oreophila C. A. Mey, an

alpine species found in the mountains of western Asia (Egorova, 1999) and C.

arctogena Harry Sm., an alpine species from North America, South America and

Eurasia, which has been treated as synonym to C. antarctogena Roiv. by Egorova

(1999) and Moore & Chater (1971). Despite its small size, the section possesses several

taxonomic problems concerning the circumscription of its species that should be firstly

resolved in order to not misinterpret posterior results from other studies such as the

following biogeographic ones. Although Carex capitata and C. arctogena are

63 ______Chapter 2. Taxonomy of the Carex capitata complex

recognized as separate species in some taxonomical treatments and online databases

(e.g. Egorova 1999; Jiménez-Mejías & Luceño, 2011; Govaerts et al., 2014), they are

considered as synonyms by other authors (e.g. Murray, 2002; http://www.tropicos.org).

Hybrids involving different members of the C. capitata complex have never been

reported.

The recognition of both taxa as different species is based on the following characters:,

C. capitata is pointed out to have generally longer inflorescences, bigger achenes,

shorter pistillate scales and smooth margins; it forms looser tussocks than C. arctogena

and occurs at lower elevations, in moist or humid areas (Table 1). Carex arctogena’s

distribution in Europe is less widespread (Fig. 2), occurring only in Scandinavian

countries. In North America, it occurs from Greenland to Mexico, where it presents a

considerable morphological and ecological variability, particularly in western North

America. Some authors (Smith, 1940; Egorova, 1999; Cayouette, 2007) stated the

necessity of more detailed studies including specimens from North America. Moreover,

C. arctogena circumscription is also unclear regarding populations found in South

America, which were thought to represent a separate species, C. antarctogena

Roivanen. As stressed by Reinhammar & Bele (2001), only a comprehensive worldwide

study of the complex using morphological, genetic and habitat variation would resolve

the systematics of these taxa.

Therefore, the aims of this study are: (i) to investigate how many taxonomical entities

are found in the C. capitata complex and their appropriate rank; and (ii) to characterize

them morphologically, ecologically and geographically.

Materials and methods

Sampled material

64 ______Chapter 2. Taxonomy of the Carex capitata complex

Owing to the scarcity of herbarium specimens for Carex capitata s.l. from South

America and the western USA, two expeditions were made to collect fresh material: one

to Patagonia (January-February 2010) and a second to the western of United States

(July-August 2010). Samples were collected for a total of 10 populations, covering the

distributional range of the species in those regions (Govaerts et al., 2014). The isotype

for C. arctogena Harry Sm. was obtained from Agriculture and Agri-Food Canada

(DAO, Ottawa) whereas high resolution scans of the holotypes and leptotypes for C.

capitata and C. antarctogena were obtained from the Linnean Society of London

(LINN) and from the University of Helsinki (H), respectively. Four hundred and forty

six herbarium specimens were obtained on loan from the following herbaria

(abbreviations according to Index Herbariorum; Thiers, 2012): A, ALA, BAA, BRY, C,

CAN, CAS, CCO, CHSC, COLO, DAO, GH, H, ICEL, M, MICH, MONTU, O, OSC,

RM, RMS, UBC, UNM, UTEP, WIN and WTU. Carex oreophila has not been studied

as its morphological circunscripcion within the section is not problematic.

Morphological study

Vegetative characters were measured using a standard rule for parts longer than 10 cm

whereas all other quantitative characters were measured to the nearest 0.1 of a

millimeter using a stereoscopic binocular Nikon microscope Olympus SZX12 and a

micrometer. Qualitative states were scored by eye. Twenty nine morphological

characters [28 quantitative (22 cuantitative and 6 discrete) and one qualitative; see Table

2], were measured on a total of 147 specimens (C. capitata, N=43; C. arctogena, N=34,

C. antarctogena, N=6; undetermined specimens N= 63). Summary statistics for all

65 ______Chapter 2. Taxonomy of the Carex capitata complex

characters including means, standard deviations and ranges were calculated for each

group in R v2.15.0 (R Development Core Team, 2011).

The lowermost, mature achene was removed from the terminal spikes of representative

samples of each group (C. capitata, C. arctogena, C. antarctogena and unclassified

specimens). The perigynium surrounding the achene was dissected away and the cell

wall of the epidermal layer of achenes was removed using a 9:1 sulfuric acid - acetic

anhydride solution in order to expose the silica bodies (Starr & Ford, 2001). Scanning

Electron Microscopy (SEM) was then employed to search for taxonomically diagnostic

micromorphological characters on the silica deposit surfaces. Samples were mounted

onto aluminum stubs with conductive carbon adhesive discs, sputter coated with a 20-25

nm layer of a gold/palladium alloy and photographed in high vacuum mode using a

Philips XL-30 ESEM with a 10kV accelerating voltage. Silica body morphology was

described according to the terminology of Schulyer (1971).

Statistical Analysis

Statistical analyses of morphological data were aimed at identifying significantly

distinct groups and diagnostic characters and to test if the studied individuals formed

different groups correlated with the different taxa traditionally recognized within the C.

capitata complex.

Histograms showing interspecific frequency differences between groups were made for

the six discrete variables. Quantitative variables for the five putative taxa were explored

using boxplots. The Shapiro Wilk normality test conducted in the data set showed that

most of the variables were not normally distributed within the putative taxa, thus

intertaxon variation was analyzed using a Kruskal-Wallis one-way ANOVA. A post-hoc

Mann-Whitney U pairwise test was also performed to assess whether differences were

66 ______Chapter 2. Taxonomy of the Carex capitata complex

significant between groups. These analyses were run in R v2.15.0 (R Development Core

Team, 2011).

Principal component analysis (PCA) is primarily used for structure detection within the

studied data, and thus, PCAs were performed to detect groups among all specimens.

These analyses were carried out using R v2.15.0 (R Development Core Team, 2011). A

first PCA was conducted using specimens of C. capitata, C. arctogena and C.

antarctogena and all 22 continuous variables. The analysis was repeated in the same

dataset using a subset of 12 quantitative variables (denoted by asterisks in Table 2) that

included the characters employed by Egorova (1999) to differentiate between C.

capitata and C. arctogena and those determined in a pilot analysis to set apart three

groups within the unclassified specimens. A correlation matrix was studied in order to

discard highly correlated variables (>0.8) within the subset (see Tables S2 and S3 in

Appendix). Although the length of the inflorescence and length of the staminate portion

were highly correlated (0.9), both were retained in the PCA because this correlation was

observed to be inconsistent in C. capitata. Consecutive PCA were performed removing

groups that were distinctly retrieved (Jiménez-Mejías & Cabezas, 2009; Valcárcel &

Vargas, 2010; Jiménez-Mejías et al., 2014).

Geography

All studied specimens were geo-referenced to determine the geographic ranges for each

putative taxon and to reveal whether taxa occur in sympatry or allopatry. Distribution

maps [from Olson et al. (2001) for world maps, and North American Commission for

Environmental Cooperation for North American maps] of all the putative taxa in the

complex were made in ESRI ArcGIS v. 9.2, using all the specimens studied.

Ecology

67 ______Chapter 2. Taxonomy of the Carex capitata complex

For the three populations collected in North America, soil pH measurements using

Cornell pH Test Kit Wide Range (Ithaca, New York) were taken to characterize habitat

conditions. A list of vascular plants associated with the complex (observed within 10 m

of a plant of C. capitata complex) was noted. Habitat characterization of all the putative

taxa in the C. capitata complex was made from field work observations and voucher

label information, unless otherwise noted.

Micromorphological study

Silica bodies are phytoliths produced by some plant species when soluble silica from the

ground water is absorbed by the roots and carried to different parts of the plant through

the vascular system. In sedges, they are found in the achene and leaf epidermal layers

(Toivonen & Timonen, 1976). Micromorphology in angiosperms has been used to

discriminate macromorphologically similar taxa (Stuessy, 2009). Silica bodies were

studied because they can sometimes show significant interspecific variation among

closely related species in Carex (Starr & Ford, 2001; Zhang, 2006). However, some

other micromorphology studies on Carex silica bodies did not help to differentiate

between closely related species (Standley, 1987; Rothrock, 1997).

Results

Raw data from all specimen measurements is available in Appendix S1. All the label

information from the herbarium specimens studied is gathered in a Botanical Research

and Herbarium Management System database

(http://dps.plants.ox.ac.uk/bol/BRAHMS/Home/Index) that is available upon request

from the author (see Figure 2).

Univariate analysis

68 ______Chapter 2. Taxonomy of the Carex capitata complex

Specimens from South America, previously identified as C. antarctogena, were

compared to specimens of C. arctogena from North America and Europe, resulted in no

statistically significant differences between them (see Table 2). Therefore, those

specimens were included in the C. arctogena group. Despite some overlap in the

measurements for many characters, all taxa present significant differences between

variables (Table 2). Based on Mann-Whitney pairwise comparisons between taxa in the

complex, the best variables to distinguish them are: culm length (overwintered or not),

the length of the staminate flowers portion, leaf length, inflorescence length and width,

perigynium width, and length of the shortest hyaline margin. All taxa can be identified

by a unique set of characters. Based on uni- and multivariate analyses, three new

taxonomic entities are identified within the studied specimens and subsequently

described. Specimens with medium-size culms, the longest pistillate scale and the

widest perigynia were referable a new species herein described as C. cayouetteana sp.

nov. (see section Species descriptions and Figure 15). Specimens with the longest

culms, leaves, inflorescence and staminate flowers portion were referable to a new

subspecies herein described as C. cayouetteana subsp. bajasierra subsp. nov. (see

section Species descriptions and Figure 16). Specimens with short culms and leaves and

the narrowest inflorescence were referable to C. cayouetteana subsp. altasierra subsp.

nov. (see section Species descriptions and Figure 17). Specimens with short culms,

small staminate flowers portion, long hyaline margins, narrow perigynia and with the

smallest staminate scales were referable to C. arctogena, which its morphological

characteristics are consistent with both its holotype and C. antarctogena holotype

(Figures 2.14, A.12 and A.13). Finally, specimens with long culms and leaves, the

widest inflorescence, the narrowest pistillate scales and the longest perigynia were

referable to C. capitata (Figure 2.13). Although discrete characters do not present

69 ______Chapter 2. Taxonomy of the Carex capitata complex

statistically significant differences between taxa (Figure A.1), in general C. arctogena

possesses more teeth along the edges of the perigynium that what is seen in other

species. For instance, C. capitata and C. cayouetteana subsp. bajasierra have smooth

perigynia or possess only a few teeth, generally no more than three, on perigynium

margins whereas C. arctogena often have between four and seven. In general, C.

cayouetteana subsp. altasierra also possess smooth perigynia, but at least one specimen

presented 16 teeth along its margins.

Multivariate analyses

During the analyses of morphological traits, one qualitative character, color of the culm

sheath, was discarded as it could not be reliably scored. Boxplots for each of the twenty

two continuous variables (Figures A.2 and A.3 in Appendix) show interspecic

differences between taxa. Only two characters (achene and leaf width) were not

significantly different among members of the complex (Table A.1 in Appendix). Mann-

Whitney pairwise comparisons reveled the following critical diagnostic characters for

differentiating between species within the complex (Table 2.8 in Appendix): length of

the longest culm overwintered (CLHMT) or from the present year (CLMH), length of

the staminate portion of the inflorescence (MSPL), leaf length (LEAFL), length of the

spike (INFLOL), width of the perigynium (PERIGW), width of the spike (INFLOW)

and length of the narrowest hyaline margin in the pistillate scale (GLUMHC). All taxa

can be identified by a unique set of characters. Specimens with the longest culms,

leaves, inflorescence and staminate flowers portion were referable to C. cayouetteana

subsp. bajasierra (Figure 2.3). Specimens with short culms and leaves and the

narrowest inflorescence were referable to C. cayouetteana subsp. altasierra (Figure

2.4). Specimens with medium-size culms, the longest pistillate scale and the widest

perigynia were referable to ‘C. cayouetteana’ (Figure 2.5). Specimens with short culms,

70 ______Chapter 2. Taxonomy of the Carex capitata complex

small staminate flowers portion, long hyaline margins, and narrow perigynia and with

the smallest staminate scales were referable to C. arctogena, being in consistence with

both its holotype and C. antarctogena holotype (Figures 2.6, A.13 and A.14 in

Appendix). Finally, specimens with long culms and leaves, the widest inflorescence, the

narrowest pistillate scales and the longest perigynia were referable to C. capitata

(Figure 2.7).

A scatter plot of the two first components in a PCA using all C. capitata and C.

arctogena specimens measured and 12 variables was made to study if they could be

clearly differentiated. As shown in Figure 2.9, there is practically no overlap between C.

capitata and C. arctogena, consisting in two distinctive clusters. PCA of the five groups

(C. capitata, C. arctogena, C. cayouetteana subsp. cayouetteana, C. cayouetteana

subsp. bajasierra and C. cayouetteana subsp. altasierra) graphically summarized the

phenetic differences among individuals. Only scatter plots from the PCA with the 12

variables are shown. Similar results are obtained when using the 22 continuous

variables measured (see Figures A.5, A.6, A.7, A.8, A.9 and A.10 in Appendix). When

comparing all specimens of C. arctogena, C. cayouetteana subsp. cayouetteana, C.

cayouetteana subsp. bajasierra and C. cayouetteana subsp. altasierra and using 12

variables, there is a clear separation of C. cayouetteana subsp. bajasierra from all the

other taxa (Figure 2.10). Then, if C. cayouetteana subsp. bajasierra is removed from the

analysis, there is a clear increase in the split of C. cayouetteana subsp. cayouetteana

from C. arctogenaand C. cayouetteana subsp. altasierra (Figure 2.11). Finally,

removing C. cayouetteana subsp. cayouetteana, the scatter plot from the PCA shows a

small overlap between C. arctogena and C. cayouetteana subsp. altasierra, although

two main clusters could be differentiated (Figure 2.12). The first two principal

component axes in PCA using 12 continuous variables, accounted for 28; 53% and 16;

71 ______Chapter 2. Taxonomy of the Carex capitata complex

7% of the total variance. . First components in a PCA using C. arctogena specimens

from North America and Europe and C. antarctogena specimens and the 12 variables

selected showed that there is not a geographical pattern within the samples (see Figure

2.8). Therefore, C. antarctogena specimens were labeled afterwards as C. arctogena.

Micromorphological characters

Silica bodies presented no significant differences between or within putative taxa in the

complex, with all the members possessing a single, circular central body in the middle

of a concave silica platform. Epidermal cell walls were commonly linear, isodiametric

and six-sided (Figure 2.15).

Geographical distribution

Carex capitata presents a circumboreal distribution and it occurs in Eurasia and in

North America (Figure 2.16). In Europe, it occurs in Iceland, Norway, Sweden, Finland,

Germany, Austria, Switzerland and Italy. In Asia, it occurs in Russia from Kola

Peninsula to Chukotka peninsula, occurring south to 50ºN in central eastern Russia.

Raymond (1949) also noted that it occurs in northern Mongolia, but no specimen from

this region was examined during this study. In North America, it occurs in Alaska, the

Yukon Territory, the Northwest Territories, British Columbia (South to ca. 50ºN),

Alberta, Saskatchewan (South to ca. 52ºN), northern Manitoba, northern Ontario and

Greenland (North to ca. 72ºN).

Carex arctogena has a bipolar and amphi-Atlantic distribution with stations in northern

Europe (Scandinavia), North America and South America (Figure 2.1). In North

72 ______Chapter 2. Taxonomy of the Carex capitata complex

America, it occurs in British Columbia (South to ca. 50ºN), northern Saskatchewan,

Manitoba (South to 52ºN), northern Ontario, northern Québec, Newfoundland and

Labrador (South to ca. 52ºN), Nunavut (until South of Victoria Island and Baffin Island,

63.5ºN), New Hampshire (White Mountains and Mt. Washington, 44ºN; Steele &

Hodgon (1973) reported to occur in Mt. Cardigan but that material was not examined)

and its northernmost latitude occurs in Greenland (North to ca. 68ºN). In South

America, it occurs in Argentinian and Chilean Patagonia, from Tierra del Fuego

(Argentina) to Neuquén province (38ºS, Argentina).

Carex cayouetteana subsp. cayouetteana, C. cayouetteana subsp. bajasierra and C.

cayouetteana subsp. altasierra are endemic to North America (Figure A.16 in

Appendix). Carex cayouetteana subsp. cayouetteana occurs only in western North

America with stations in Colorado, Utah, Montana, Wyoming, Nevada, California,

Washington, Alberta and British Columbia (North to ca. 49ºN). C. cayouetteana subsp.

bajasierra occurs only in northern California and southern Oregon (Deschutes, Jackson

and Lake Counties). C. cayouetteana subsp. altasierra is a Californian endemic,

restricted to high elevations in the Sierra Nevada (Inyo, Mono, Tulare and Tuolumne

Counties).

Ecological requirements

Carex capitata is an alpine species. In northern latitudes such as Alaska or the European

Arctic, it is found in tundra and taiga (boreal forest) environments whereas in southern

latitudes, such as central Europe or western Canada, it is found in alpine or subalpine

areas. It occurs in rich and calcareous fens, mires, peat-bog margins, meadows, wet

tundra and other humid or moist habitats, sometimes with moss as also noticed by Smith

73 ______Chapter 2. Taxonomy of the Carex capitata complex

(1940). In Alaska, it is also found in marshes and poplar forest from lowlands (400 m)

to at least 800 meters. In northeastern North America, it is mainly found in areas

adjacent to Hudson Bay, rare or local in alpine summits towards South. Carex capitata

elevational occurrence in Italy is at 1900 - 1980 m (in the South Tyrols). It has been

reported to be strictly a calciphile or calciphilous (Smith, 1940; Nilson, 1991;

Cayouette, 2007), but such information was not taken from the label data from the

specimens examined for this study.

Carex arctogena is an arctic-alpine species. It generally occurs in wind-exposed alpine

heaths, often dominated by Empetrum (Ericaceae) and also in cliffs, ridges, summits

and in dry areas often dominated by rocky or gravelly soil. In northeastern North

America it is found locally in New Hampshire (Alpine Garden and Mt. Cardigan) at

1900 m, one of the highest elevations within its entire distribution together with its

southernmost localities in British Columbia (ca. 2000 m). Similarly, it occurs near this

altitude in northern Patagonia (Neuquén). In southern South America, it occurs in humid

areas such as bogs, wet meadows and eutrophic marshes at low elevations, often in

areas of high floral diversity (Table 2.2). In the southernmost region of Patagonia,

Tierra del Fuego, C. arctogenawas found in a semi-humid grassland, dominated by

tufted grasses interspersed with Empetrum rubrum at low elevations (Table 2.2). It has

been reported to grow in either calciphile, peridotite, gneiss, granite or serpentine soils

(Smith 1940; Nilson 1991) but such information is generally missing in voucher

specimens.

Carex cayouetteana subsp. cayouetteana generally occurs in acidic and rocky soils (see

Table 2.3), in wind-exposed, alpine moist tundra areas and sometimes in dry meadows.

It is found from ca. 2000 m in Washington and California to at least 3500 m in

Colorado and Utah, where it can grow on quartzite soils.

74 ______Chapter 2. Taxonomy of the Carex capitata complex

C. cayouetteana subsp. bajasierra may occur in acidic-neutral pH soils (see Table 2.4),

in wet meadows or mires surrounded by woods. In northern California (Tehama, Plumas

and Butte Counties) and southern Oregon (Lake and Jackson Counties), it occurs at

unusually low elevations for the complex at this latitude (ca. 1400 m). In Sierra and El

Dorado Counties, it occurs in wet marshy meadows and in open Pinus contorta forests

at ca. 1980- 2300 m where it reaches its highest elevation.

C. cayouetteana subsp. altasierra is restricted to the highest elevations in California. It

occurs in non-glaciated plateaus and wet banks. On the North side of Mount Humphries

(‘Humphries Plateau’, Inyo Co.) it grows at 3900 m and also at 3600 m at Mono Mesa

(Inyo Co.); in northeastern Tulare Co., in wet banks at ca. 3400 m. It is found in

Tuolumne Co., in soil formed from metamorphic rocks, in non-glaciated areas at ca.

3800 m

Discussion

There are geographical and ecological differences between the taxa found in this

complex: C. capitata presents a circumboreal distribution whereas C. arctogena

presents a bipolar distribution; Carex cayouettena subsp. cayouetteana is only found in

western North America from (2000 to 3800 m); C. cayouetteana subsp. bajasierra is

restricted to southern Oregon and northern California (1400 - 2300 m); and C.

cayouetteana subsp. altasierra occurs locally at high elevations (3400 - 3900 m) in

California. Only 8 herbarium specimens of C. cayouetteana subsp. altasierra were

observed during the course of this study. Giving the extreme elevation at which this

species occurs, this might indicate that C. cayouetteana subsp. altasierra is rare or that

the habitat in which it occurs has been under collected.

75 ______Chapter 2. Taxonomy of the Carex capitata complex

Although C. antarctogena specimens from South America displayed statistically

significant morphological differences regarding to northern hemisphere specimens of C.

arctogena in some variables, they are not enough to differentiate between northern and

southern specimens. These differences are of a 10% in average and for one variable,

length of the staminate portion is of 38% (but this variable has a standard deviation of

the same order of magnitude as the mean). Thus, C. antarctogena and C. arctogena are

treated as synonyms in this study, which is consistent with the previous morphological

analysis by Moore & Chater (1971). In congruence, in Chapter 3, molecular analyses of

three chloroplast regions (matK, atpF-atpH and rps16; 2297 characters), show no

genetic differences between C. arctogena from the Northern vs. Southern Hemisphere.

On the other hand, some variability was found within C. capitata specimens from

Russia, Austria, Ontario and Alberta, which have culms longer than 41 cm long. This

plasticity within C. capitata was also remarked by Raymond (1949) in some specimens

collected in Québec (Lac De l’Ours). However, in our opinion, this appears to be no

more than a regional trend and it is not correlated with other morphological characters,

so it might not deserve taxonomical recognition. All taxa in the C. capitata complex are

long-lived perennials, wind-pollinated and reproduce sexually. Hybrids between

members of the complex have not been reported.

A large number of new species has been described over the last few decades in North

America North of Mexico (Ertter, 2000). This is especially true for the genus Carex

(Naczi, 1993; Naczi et al., 1998; Saarela & Ford, 2001), with an average of two taxa

described per year over the last 20 years (Ertter 2000). This rich biodiversity in North

America could be due to ecological diversity and historical environmental

transformations due to paleoclimatic changes, especially during Pleistocene climatic

oscillations, as it has been highlighted in many intensive Flora studies (e.g. Ball et al.,

76 ______Chapter 2. Taxonomy of the Carex capitata complex

2002) and should continue to be the focus of research interests in order to better

understand species distributions.

All five species of the C. capitata complex occur in North America and the three new

taxa are endemic to North America, which provides further evidence of the taxonomic

richness that exists within North American Carex. Some other examples include C.

maritima Gunn. species complex, a bipolar species widely distributed in North America,

whose ecological and morphological variability led Kreczetowitcz (1932) to described

twelve different species and some other botanists to describe new taxa [e.g. C.

incurviformis Mack. varieties, C. maritima var.setina (Christ) Fernald or C. maritima

var. misera (Kük.) Fernald]; and forms [e.g. C. maritima f. inflata (Simmons) Polunin].

It is also remarkable the studies made by Naczi et al. (2002) who described seven new

Carex species from North America (C. acidicola Naczi, C. calcifugens Naczi, C.

paeninsulae Naczi, E. L. Bridges & Orzell, C. thornei Naczi, C. kraliana Naczi &

Bryson, C. gholsonii Naczi & Cochrane and C. infirminervia Naczi).

77 ______Chapter 2. Taxonomy of the Carex capitata complex

Taxonomic treatment

The following key helps to identify the species of C. capitata complex recognized in

this study. Mature, complete and well developed specimens are necessary for correct

identifications.

Key to species of the C. capitata complex

1. Tallest culm < 160 mm long, inflorescence < 3 mm wide…. C. cayouetteana subsp. altasierra .(Y3) 1. Tallest culm > 160 mm long, inflorescence > 3 mm wide……………………….2

2. Inflorescence > 13 mm long…………………………………C. cayouetteana subsp. bajasierra. (Y2)

2. Inflorescence < 13 mm long……………………………………………….…3

3. Perigynia < 17 mm wide with more than 3 teeth, hyaline margins on the pistillate scales in a triangular shape…...………………. C. arctogena

3. Perigynia > 17 mm wide with less than 3 teeth, hyaline margins on the pistillate scales in an inverted V shape…………………………………..4

4. Tallest culm < 230 mm long, staminate portion > 2.4 mm long, pistillate scales > 2.2 mm long and > 1.5 mm wide .....C. cayouetteana subsp. cayouetteana. (Y)

4. Tallest culm > 230 mm long, staminate portion < 2.4 mm long, pistillate scales < 2.2 mm long and < 1.5 mm wide .....C.capitata

78 ______Chapter 2. Taxonomy of the Carex capitata complex

Species descriptions

Carex capitata L., Syst. Nat. (1759) 10th ed., 2:1261. Type: Sweden, Lapponia. Leg.

Solander. Stockholm Linnean Herbarium 378.13 (S-LINN: IDC 378.13) photo!

Herb forming loose to dense tussocks. Roots dark yellow, light brown, yellow or

greyish-yellow. Culms 12-49 cm high and 0.6-1.0 mm wide at the middle, slender, wiry,

more or less dentate on the margins and mainly near the apex. Leaves usually 3-5 per

culm, old leaves persistent, most often shorter than the culm; leaf sheaths dark orange-

brown, dark brown-red or dark brown at the base, sparingly filamentose.; blades 11.5-36

cm long and 0.4-1.5 mm wide in the middle, filiform, stiff, erect or recurving, truncate

mouth; ligule very short, obtuse. Spike solitary, androgynous, globose, ovoid or

trigonal, with staminate portions covering 15 % to 34 %, fairly densely packed, of 5.5-

10.3 mm long and 3.3-5.4 mm wide, staminate portions from 0.8-3.5 x 0.5-1.3 mm, 5-

15 staminate flowers, 12-27 pistillate flowers; bract absent or rarely present; staminate

scales erect, obovate or ovate, the body orange, dark yellow-orange or brown-yellow

with hyaline margins located in the distal 1/3 and 0.1-0.2 mm wide, often folded,

glabrous, 1.6-2.9 x 0.6-1.5 mm, incomplete veins, acute apex; stamen with anthers 1.0-

1.5 mm long; pistillate scales ovate or broadly ovate, the body orange, dark orange or

brown, hyaline margins absent or 0.1-0.5 mm in the central portion and 0.1-2.25 mm

along the edges, central nerve rarely present, glabrous, 1.5-2.5 x 0.8-1.8 mm, shorter

than the perigynium body and reaching 1/2 of perigynia body length, narrower than the

perigynia and sometimes not reaching 3/4 of perigynia width; distal perigynia erect or

ascending, mostly spreading or the lowermost descending or retracted in the proximal

part, the body greenish-light yellow in proximal half, yellow to yellow-grayish or brown

with some redness in the distal half, surface often shiny with some red dots, 1.8-3.6 x

1.3-2.2 mm, margins sometimes winged especially in the 1/3 proximal and 0.1-0.2 mm

79 ______Chapter 2. Taxonomy of the Carex capitata complex

wide, smooth (rarely 0-5 teeth), round bases, apex acute, contracting gradually into a

beak; beaks brown or orange-brown, apex orange or hyaline, 0.8-1.7 mm long x 0.2-0.3

mm wide at the base, straight; gynoecium with 2 stigmas; rachilla often visible in relief

on the side of abaxial perigynia, setaceous, as long or slightly longer than achenes;

achenes ellipsoid, broadly ellipsoid or almost orbicular, the body light yellow, yellow-

greenish, non-glossy surface, 1.1-2.1 x 1.0-1.5 mm, filling more than 2/3 to 3/4 of the

perigynium, broadly cuneate or rounded at the base, apex acute, obtuse or truncated;

style bases absent or persistent by the bottom of the style.

Notes: C. capitata is easily differentiated from other members of the complex by its

spreading perigynia with the lowermost sometimes even descending, similar to the

morphological condition separating the species pair C. typhina Michaux and C.

squarrosa L. It can also be easily recognized from all other members of the complex by

its pistillate scales, shorter and narrower than the perigynia; its small staminate portion;

the presence of some redness in the perigynia and glabrous perigynia.

Distribution: Europe (ICE, NOR, SWE, FIN, GER, AUT, SWI, ITA); N Russia; N

North America (ASK, YUK, NWT, BRC, ABT, SAS, MAN, ONT, GNL).

Ecology: Tundra, taiga (boreal forest) and alpine and subalpine areas, in various humid

soils (fens, mires, meadows).

Carex arctogena Harry Sm., Acta Phytogeogr. Suecica (1940), 13:193. Type: Sweden,

Torne Lappmark, karesuando, Moskana ca. 1000 m.s.m. 26/7 1933, Harry Sm. (UPS

Holotype) photo! (Fig. A.13 in Appendix)

List of specimens studied: Argentina, Dept. Chos Malal, 2300 m, Boelcke, O., Correa,

M.N.; Bacigalupo, N.M., 30.1.1964, (BAA, 11368). Dept. Chos Malal, 2300 m,

Boelcke, O., Correa, M.N.; Bacigalupo, N.M., 30.1.1964, (BAA, 11368).A, Mendoza,

80 ______Chapter 2. Taxonomy of the Carex capitata complex

Cordillera del Rio Barrancas, Kurtz, F., 16.11.1888, (MICH). Canada, Alberta, Mercoal,

Rousseau, J., 18.7.1947, (COLO, 13811). Alberta, Mercoal, 4300 ft, Malte, M.O.,

Watson, W.R., 8.8.1925, (RM, 280606). British Columbia, Pine Pass, 1402 m, Argus,

G.W., 12.7.1973, (CAN, 372267). British Columbia, 7228 ft, Calder, J., 149035,

Parmelee, J.A.; Taylor, R.L., 8.8.1956, (COLO, 149035). British Columbia, Mount

Apex, 7100 ft, Calder, J., Savile, O., 11.8.1953, (RM, 252249). Manitoba, Fort Chimo,

Rousseau, J., 14.8.1951, (WIN, 22355). Manitoba, Baralzon Lake, Scoggan, H.J.,

22434, Baldwin, W.K.W., 28.7.1950, (WIN, 22434). Manitoba, Hudsons Bay Co.,

Duck Lake, Scoggan, H.J., Baldwin, W.K.W., 10.8.1950, (WIN, 22435). Manitoba, Fort

Chimo, Legault, A., 22.7.1963, (COLO, 491481). Manitoba, Hudsons Bay Co., Duck

Lake, Scoggan, H.J., Baldwin, W.K.W., 10.8.1950, (CAN, 201506). Manitoba,

Baralzon Lake, Scoggan, H.J., Baldwin, W.K.W., 30.7.1950, (CAN, 202500).

Manitoba, Nueltin Lake, Baldwin, W.K.W., 26.7.1951, (CAN, 212816). Manitoba,

Cochrane River, Baldwin, W.K.W., 3.7.1951, (CAN, 212817). Manitoba, Baralzon

Lake, Scoggan, H.J., Baldwin, W.K.W., 28.7.1950, (CAN, 201507). Newfoundland-

Labrador, Esker area, 838 m, Mäkinen, Y.,Kankainen, E., 21.7.1967, (CAN, 314758).

Canada, Newfoundland-Labrador, Twin Falls, Hustich, I., 6.7.1967, (CAN, 313311).

Nunavut, Upper Hood River, 100 m, Gould, W..7.1995, (COLO, 475773). Ontario,

Kenora District, Patricia Portion, Riley, J.L., 12.8.1980, (CAN, 462937). Ontario,

Hudson Bay Lowlands, Porsild, A.E., Baldwin, W.K.W., 4.7.1957, (CAN, 278707).

Quebec, Fort Chimo, Sørensen, T., 17.8.1959, (C). Quebec, Baie d'Ungava, Blondeau,

M., 1.8.1993, (WIN, 53902). Quebec, Baie d'Ungava, Rousseau, J., 23.7.1951, (WIN,

22356). Quebec, Lac Jaucourt Region, Lichteneger Lake, 487 m, Argus, G.W.,

16.7.1974, (CAN, 3779977). Quebec, Boatswain Bay, Baldwin, W.K.W.,Hustich, I.;

Kucyniak, J.; Tuomikoski, R., 8.7.1947, (CAN, 17333). Quebec, Lac Payne, Legault,

81 ______Chapter 2. Taxonomy of the Carex capitata complex

A., 2.8.1965, (CCO, 23398). Quebec, Northern Quebec, Lake Payne, Legault, A.,

210789, Brisson, S., 2.8.1965, (COLO, 210789). Quebec, Ungava, Husons Bay, Dutilly,

A., 233644, Lepage, E., 21.3.1945, (RM, 233644). Quebec, Fort Chimo, Calder, J.,

31.7.1948, (RM, 255325). Quebec, Hudson Bay, Cairn Island, Abbe, E.C., Abbe, L.B.;

Marr, J., 30.7.1939, (RM, 252521). Quebec, Hudson Bay, Great Whale River, Calder,

J., Savile, O. Kukkonen, I., 8.8.1959, (RM, 260486). Quebec, Lac Kopeteokash,

Rousseau, J., 18.7.1947, (RM, 228636). Saskatchewan, Vicinity of Patterson Lake,

Argus, G.W., 20.7.1963, (CAN, 282691). Saskatchewan, Northeastern Saskatchewan,

Patterson Lake, Argus, G.W., 20.7.1963, (RM, 277437). Finland, Enontekiö,

Kilpisjärvi, Saana, 750 m, Roivainen, L., 8.7.1935, (H, 127310). Enontekiö, Kilpisjärvi,

Saana, 750 m, Väre, H., 29.7.2004, (H, 805587).A, Enontekiö Lapland, 825 m, Väre,

H., 17.7.2006, (H, 809948). Inari, Vätsäri Wilderness Area, Kulmala, H., 27.7.1996, (H,

717201). Lapponia Imandrae, Lindén, J., 18.7.1891, (H, 325665). Lapponia Imandrae,

Axelson, W.M., Borg, V., 24.7.1901, (H, 325667). Lapponia murmanica, 550 m,

Brotherus, V.F., .8.1887, (H, 325639).A, Petsamo, Cajander, A., 10.7.1927, (H,

325644). Porojärvet, Toskalhar, 950 m, Roivainen, H., Ollila, L., 15.7.1955, (H,

127313).A, Porojärvet, Toskalhar, 910 m, Roivainen, H., 15.7.1966, (H, 179889).

Foutell, C.W., Jalan, M.J., 10.8.1899, (H, 325657). Germany, Altevatn, 500 m,

17.8.1967, (M, 0151943). Greenland, Arfersiorflk, Itjvdljarssuk, 75 m, Fredskild, B.,

Dalgaard, V., 19.7.1987, (COLO, 456814). Groenlandia meridionalis, Kangerdluarssuk,

Hansen, C., Hansen, K.; Petersen, M., 4.7.1962, (CAN, 282521). Nigerdleq, Jørgensen,

L.B., 15.7.1966, (CAN, 311369). Vestgrønland, Pingorssuaq Kitdleq, 400 m, Hanfgarn,

S. 3, 11.8.1983, (C). Tugtilik Lake, 10 m, Elsley, J.E. 15.8.1967, (M, 0151948).

Lagerkranz, J., 2.8.1936, (RMS, 153944). Norway, Finnmark, Sör-Varanger, Bugöynes,

Toivonen, H., 30.7.1971, (H, 1081734). Finnmark, Sör-Varanger, Bugöynes, Toivonen,

82 ______Chapter 2. Taxonomy of the Carex capitata complex

H. 30.7.1971, (H, 1081733). Nordland, Narvik hd., Skjomen, Skifte, O., GRaff, G.;

Spjelkavik, S., 11.8.1973, (H, 1679404). Norland, Sulitjelma, Skifte, O., 1.8.1962,

(DAO, 285800). Sverige, Abisko, Paddas, Lid, J., 1300264, 2.8.1950, (H, 1300264).

Troms, Bardu, Leinavatn, 498 m, Engelskjøn, T., Engelskjøn, E.M., 7.7.1977 (C).

Troms, Bardu, Altevatn, 580 m, 18.8.1967, (M, 0151942). Troms, Bardu, Kampaksla,

780 m, Engelskjøn, T., Skifte, O., 9.8.1978, (H, 1685049). Russia, Petsamo, Petchenga,

Vouvatusjärvi, Piirainen, M., 27.7.1995, (H, 1682990). Sweden, Torne Lappmark,

Karesuando, 1000 m, Smith, H., 26.7.1933, (DAO, 257429). Torne Lappmark,

Karesuando, 1000 m, Smith, H., 26.7.1993, (H, 1652844). Torne Lappmark, Jukkasjärvi

parish, 550 m, Alm, G., Smith, H., 23.7.1939, (H, 1300259). USA, New Hampshire,

Coos Co., Mt. Washington, Hodgon, A.R., Gale, M., 30.6.1950, (DAO, 257427). New

Hampshire, White Mountains, Mt. Washington, Forbes, F., 9.8.1902, (RMS, 242089).

New Hampshire, Alpine Garden, Mt. Washington, Sargent, F.H., 5.7.1942, (BRY,

143916). New Hampshire, Alpine Garden, Mt. Washington, 5000 ft, Löve, A., Löve, D.,

27.7.1958, (COLO, 288736). New Hampshire, Alpine Garden, Mt. Washington, Löve,

A., Löve, D., 3.7.1960, (COLO, 295019). New Hampshire, White Mountains, Mt.

Washington, Forbes, F., 9.8.1902, (RM, 50212). New Hampshire, White Mountains,

Mt. Washington, Eggleston, W.W., 29.7.1899, (RM, 44595). New Hampshire, Alpine

Garden, Mt. Washington, Eggleston, W.W., 29.7.1899, (RM, 23379). New Hampshire,

White Mountains, Faxon, C.E., 1.9.1877, (CAN, 162720).

≡ C. capitata L. ssp. arctogena (Harry Sm.) Hiit., Luonnon YstЉvЉ 48: 52-64. (H)

photo! (≡ C. capitata L. ssp. arctogena (Harry Sm.) Böcher, in Medd. om Grønl.147(9),

1952. Isonym)

83 ______Chapter 2. Taxonomy of the Carex capitata complex

≡ C. capitata L. var. arctogena (Harry Sm.) Hultén, Kungl. Sv. Vet. Ak. Handl. (1958),

4 (7):38. Uppsala. Type: Sweden, Torne Lappmark, Karesuando, Moskana ca. 1000

m.s.m. 26/7 1933, H. Smith. (UPS) photo!

≡ C. capitata f. arctogena Raymond, Contrib. bot. Univ. MontrЋal (1949), 64:38.

= C. capitata f. alpicola Andersson, Bot. Not. (1849), 2.

= Carex antarctogena Roiv., Ann. Soc. Zool. Bot. Fenn. Vanamo (1954), 28 (2): 197-

198. Type: [Chile, Tierra del Fuego] Estancia Vicuña, in palude. H. Roivanen (H

Holotype) photo!

Carex rahuiensis Kurtz. ex. Kükenth., Bot. Jahrb. (1900), 27:495 - nomen nudum,

according to Smith (1940).

Kurtz based Carex rahuiensis on plants he collected in Argentina (Kükenthal 1900).

According to Smith (1940) it is nomen nudum, although he did not see any specimen

from Kurtz’s collections. Carex antarctogena was described by Roivanen on the basis of

Argentinian specimens which he considered to be more robust in structure and to have a

greater number of staminate flowers and perigynial teeth than C. arctogena from the

northern Hemisphere. The present morphological study does not support these

observations since specimens from South America are not statistically significant bigger

than the North American or European specimens (Table 2.7). Both species will

therefore be considered as synonyms here. In Chapter 3, molecular analyses show no

genetic differences between C. arctogena samples from the northern vs. southern

Hemisphere in the three chloroplast regions and five nuclear loci studied.

Herb forming loose to dense tussocks. Roots sometimes short-creeping, yellow or

reddish. Culms 10-33 cm tall and 0.5-1.1 mm wide in diameter at the middle, slender,

84 ______Chapter 2. Taxonomy of the Carex capitata complex

wiry, more or less dentate on the margins and dense towards the apex. Leaves erect, 3-5

per culm, old leaves persistent, shorter or as long as the culm; leaf sheaths dark red or

brown at the base, sparingly filamentose; blades 9-29 cm and 0.4-1.0 mm wide at the

middle, filiform, stiff, erect or recurving, truncate mouth; ligule very short, obtuse or

nearly truncate. Spike solitary, androgynous, globose, ovoid or trigonal, with staminate

portions covering 20 % to 37 %, fairly densely packed, 5.2-9.8 mm long x 2.9-4.7 mm

wide, staminate flower portion 1.2-3.7 mm long x 0.5-1.3 mm wide, pistillate flowers

portion 3.5-6 mm long, 5-26 staminate flowers, 9-32 pistillate flowers; bract absent,

rarely present; staminate scales erect, obovate, broadly obovate or ovate, the body

yellow or olive-brown with hyaline margins located in the distal 1/3, 0.1-0.2 mm wide,

often folded, glabrous, 1.0 to 2.8 mm long x 0.7 to 1.6 mm wide, with 1-3 veins, apex

acute; stamens with anthers 0.6-1.4 mm long; pistillate scales ovate or broadly ovate,

the body yellow, orange-brown or dark brown with hyaline margins rarely absent and

typically occupying the proximal and distal portions, length of 0.1-1.0 mm in the central

portion and 0.4-2.6 mm along the edges in a triangular shape, no nerve or one,

incomplete, glabrous, 1.0-2.6 x 1.4-3.0 mm shorter than the perigynia and reaching 3/4

of perigynia body length or until the base of the beak, wider or little narrower than

perigynia; distal perigynia erect or ascending, proximal mostly spreading, the body

greenish or yellow on the proximal half and dark grayish, yellow-green or brownish

green in the distal half, surface glossy, 1.5-3.2 x 1.0-2.0 mm, 0.8-1.4 mm, margins

sometimes winged especially in the proximal half and 0.1-0.3 mm wide, almost always

scabrous (1-16 teeth), cuneiform base, abruptly contracted into a beak; beak brown,

dark-brown or olive-brown, apex orange or hyaline, 0.3-0.9 mm long and 0.2-0.3 mm

wide at base, mostly straight, bifid; gynoecium with 2 stigmas; rachilla often visible in

relief on the side of abaxial perigynia, setaceous, as long or slightly longer than the

85 ______Chapter 2. Taxonomy of the Carex capitata complex

achene; achenes ellipsoid, broadly ellipsoid or almost orbicular, the body greyish,

yellow or dark, non-glossy surface, 1.4-1.9 mm long x 0.7-1.7 mm wide, filling more

than 3/4 of the perigynium, broadly cuneate or rounded at the base, apex obtuse or

truncated; style bases absent or persistent by the bottom of the style.

Notes: C. arctogena is differentiated from all other members of the complex by its

pistillate scales, broader and as long or longer than the perigynia; its scabrous perigynia,

and its hyaline margins along pistillate scales, which have a triangular shape and which

can cover up to half of the surface of the scale. It is most similar to C. cayouetteana

subsp. cayouetteana and C. cayouetteana subsp. altasierra but they can be easily

separated by the characters mentioned above.

Distribution: Europe (NOR, SWE, FIN); N Russia; N North America (NUN, BRC,

SAS, MAN, ONT, QUE, NFL, GNL, NWH) and S South America (AGS, CLC).

Ecology: Arctic-alpine areas and wind-exposed alpine heaths, in soils with low water

content.

Carex cayouetteana subsp. cayouetteana

Holotype: Canada, Alberta: Banff National Park, Snow Creek Pass, A.E. Porsild 22673

(CAN-266077).

Paratype: USA, Colorado: Clear Creek Co., Loch Lomond, W.A. Weber, T. Koponen &

P. Nelson s.n. (CAN-374041).

Herb forming loose tussocks. Roots light brown to yellowish. Culms 11-26 cm tall and

0.6-1.1 mm in diameter at the middle, slender, wiry, more or less dentate on the margins

and dense towards the apex. Leaves 3-6(7) per culm, old leaves persistent, shorter than

culm; leaf sheaths yellow or light brown at the base, sparingly filamentose; blades11-19

86 ______Chapter 2. Taxonomy of the Carex capitata complex

cm long and 0.4-0.9 mm wide in the middle; ligules obtuse or nearly truncate. Spike

solitary, androgynous, trigonal to ovoid, lanceolate, with staminate portions wider at the

bottom and covering 50 % to 60 %, densely packed, of 6.1-12.8 mm long x 3.5-6.1 mm

wide, staminate portions from 0.9- 6.7 x 1.5-2.9 mm, pistillate 3.9-6.4 mm long, 15-26

staminate flowers, 17-32 pistillate flowers; bract absent or rarely when present;

staminate scales erect, broadly obovate or ovate, the body dark brown or yellowish,

central bands are not clearly delineated with hyaline margins located in the 1/3 distal

and 0.1-0.15 mm wide, often folded, glabrous, 1.8-3.0 x 0.6-1.9 mm, with 1-2 veins,

apex acute, subacute or rounded; pistillate scales ovate or broadly ovate, the body dark

brown, hyaline margins absent or occupying the distal portions, length of 0.1-1 mm in

the central portion and 0.1-0.5 mm crossing the edges, one nerve clearly marked and

surrounded by light brown or light yellow, glabrous, 1.5-3.4 x 1.2-2.5 mm shorter than

the perigynia and reaching full or 3/4 of body length perigynia, wider or as wide as

perigynia, apex rounded, truncated or obtuse; perigynia distal erect or ascending, most

proximal spreading, the body greenish-yellow in proximal part, dark brown to brown in

the half distal until top of the achene, surface gloss with some redness, 1.5-3.4 x 1.2-2.5

mm, 0.6-1.5 mm, margins sometimes with nerves, almost scabrous (0-5(7) teeth),

obtuse angle at the bottom, acute apex contracted smoothly into a beak; beak dark

brown, apex orange or hyaline, long of 0.9-1.9 mm, mostly straight, teeth acuminate,

bifid, smooth; gynoecium with 2 stigmas; rachilla often visible in relief on the side of

abaxial perigynia, setaceous, as long as or slightly surpassing; achenes ellipsoid, broadly

ellipsoid, the body dark yellow to light brown, glossy surface, 1.0-2.3 x 0.6-1.8 mm,

covering over 3/4 volume perigynia, broadly cuneate, rounded or rotund at the base,

apex obtuse or truncated, wrinkled; beaks marked by the straight base of the style.

87 ______Chapter 2. Taxonomy of the Carex capitata complex

Notes: C. cayouetteana subsp. cayouetteana can be identified by its staminate portion,

as long as the pistillate portion, presenting a cone shape; pistillate scales, broader and as

long as the perigynium beak, or longer than the perigynia; its scabrous perigynia,

usually with 2-3 teeth; its hyaline margins in the pistillate scales, which are around 1

mm wide and go around the edges of the scale, drawing an inverted V shape; its brown

perigynia beak and green perigynia body. Carex arctogena can be easily separated from

C. cayouetteana subsp. cayouetteana by its staminate portion, shorter and cylindrical in

C. arctogena; its hyaline margin with triangular shape; for having more teeth in the

margins of the perigynium and less number of perigynia in the spike.

Distribution: North American endemic (COL, UTA, WYO, NEV, CAL, WAS, ALB,

BRC).

Ecology: Tundra and alpine areas, in dry, acidic and rocky soils. 2000 - 3500 m.

Etymology: This taxon is named after Jacques Cayouette, a passionated botanist who

has spent his life working extensively in North American sedges and particularly in

Québec.

C. cayouetteana subsp. bajasierra

Holotype: USA, California: Butte Co., near Cherry Hill Campground, Lassen National

Forest, J. Starr 10S-054 & T. Villaverde.

Paratypes: USA, California: El Dorado County, Lake Tahoe Basin Management Unit,

J.R. Starr & J. Thibeault 07-44 (CAN). California: Sierra County, Tahoe National

Forest, J.R. Starr & J. Thibeault 07-52 (CAN). California: Butte Co., Lassen National

Forest, Forest Ranch, Cheery Hill meadows, near Cherry Hill campsite. J.R. Starr & J.

Thibeault 06016 (CAN). California: Sierra Co., Yuba Pass-Weber Lake Road, V.H.

88 ______Chapter 2. Taxonomy of the Carex capitata complex

Oswald & L. Ahart 8221 (CHSC-66824). California: Tehama Co., L. Ahart 13.051

(CHSC-94326). Oregon: Deschutes Co., C. Halpern 600 & T. Magge (OSC-159046).

Herb densely caespitose. Roots light brown, orange or dark yellow. Culms 19-54 cm tall

and 0.7-1.0 mm at the middle, slender greenish or yellowish at the base. Leaves 3-5 per

culm; leaf sheaths dark brown to light brown at the base, sparingly filamentose; blades

13-27 cm long and 0.5-0.9 mm wide in the middle; ligules, acute, obtuse or nearly

truncate. Spike solitary, androgynous, trigonal, slender, with staminate portions

covering 50 % to 70 %, loosely packed, of 6.8-16.9 mm x 3.2-4.6 mm, staminate

portions 2- 10.5 x 1-1.5 mm, pistillate 3.5-6 mm long, 30-37 staminate flowers, 8-

15(30) pistillate flowers; bract absent or rarely when present; staminate scales erect,

obovate, broadly obovate or ovate, the body light brown to light yellow in the middle

portion, central bands are clearly delineated, hyaline margins located in the 1/3 distal

and 0.1-0.25 mm wide, often folded, glabrous, 1.6-2.9 x 0.8-1.8 mm, 1 vein, apex

truncate or rounded; stamen with anthers 2-2.6 mm long; pistillate scales ovate or

broadly ovate, the body brown to light brown, orange towards the edges with hyaline

margins absent or occupying the proximal and distal portions, length of 0.1-0.3 mm in

the central portion and 0.1-1.8 mm crossing the edges, one nerve marked, glabrous, 1.2-

2.7 x 1.4-2.2 mm longer, sometimes as long as or shorter than the perigynia, reaching

3/4 of body length perigynia, wider and the bottom, a little narrower or about the same

width as perigynia in the distal portion; perigynia distal erect or ascending, proximal

spreading, the body greenish yellow in 1/2 proximal, dark brown or light brown in the

half distal surface, gloss stinks, 1.5-3.1 x 1-2 mm, 1.2-2.2 mm body length perigynia,

almost always smooth (0-3 teeth), base subacute or rounded, apex contracted smoothly

into a beak; beak brown to dark brown, apex orange or hyaline, mostly straight, teeth

acuminate, bifid; gynoecium with 2 stigmas; rachilla often visible in relief on the side of

89 ______Chapter 2. Taxonomy of the Carex capitata complex

abaxial perigynia, setaceous, as long as or slightly surpassing, 1.2-1.9 x 1 mm; achenes

ovoid or almost orbicular, the body light brown, glossy surface, 1-2 x 0.5-1.4 mm,

covering over 3/4 volume perigynia, rounded at base, apex obtuse or truncated; beaks

absent or marked by the straight base of the style.

Notes: C. cayouetteana subsp. bajasierra is easily differentiated by its staminate

portion, usually longer than the pistillate portion; its perigynia ascending, loosely

packed; its long culms, much longer than the leaves. It occurs in wet meadows at low

elevations in California.

Distribution: Western North American endemic (CAL, ORG).

Ecology: Wet meadows in boreal areas, in soils with high water content. 1400 - 2300 m.

C. cayouetteana subsp. altasierra

Holotype: USA, California: Tulare Co., Sierra Nevada, Army Pass, J.T. Howell s.n.

(DAO-257423).

Paratypes: USA, California: Inyo Co., Mono Mesa, J.T. Howell 22750 (WTU-137524).

California: Northeastern Tulare Co., Sierra Nevada, Central Basin, Lower lake, P.A.

Munz 12669 (WTU-133536).

Herb forming loose to dense tussocks, caespitose. Roots light yellow or light brown.

Culms 8-20 cm tall and 0.7-1.0 mm in diameter at the middle. Leaves 3-4(7) per culm,

90-290 x 0.4-1.0 mm; leaf sheaths brown-dark red or orange, sparingly filamentose;

blades 8-14 cm long and 0.4-0.9 mm wide in the middle; ligules, acute, obtuse or nearly

truncate. Spike solitary, androgynous, ovoid or trigonal, with staminate portions

covering 35 % to 60 %, densely packed, of 6.2-8.5 mm x and 2.5-4.0 mm, staminate

portions from 2.2-4.9 x 1.2-1.7 mm, pistillate 2.0-4.2 mm long, 26-30 staminate

90 ______Chapter 2. Taxonomy of the Carex capitata complex

flowers, 14-16 pistillate flowers; bract absent or rarely when present; staminate scales

erect, obovate, broadly obovate or ovate, the body pale yellow, light brown to dark

brown, central bands are clearly delineated with hyaline margins located in the 1/3 distal

and 0.1-0.2 mm wide, often folded, glabrous, 1.9-2.5 x 0.9-1.2 mm, with 1 vein, apex

acute to subacute; pistillate scales the body dark brown, light brown to orange towards

the edges, hyaline margins occupying the proximal and distal portions, length of 0.1-0.5

mm in the central portion and 0.1-2.0 mm crossing the edges, central nerve rarely

present, ovate or broadly ovate, glabrous, 1.8-2.1 x 1.15-2.0 mm, as long as or shorter

than the perigynia and reaching 3/4 of body length perigynia until the base of the beak,

wider or as wide as perigynia in the bottom and narrower than perigynia in the distal

part, subacute apex, scarbid; perigynia erect or ascending in the distal part, mostly

spreading in the proximal part, the body greenish or light yellow in 1/2 proximal part

with some redness, dark brown in the half distal portion, not very gloss surface, 2.0-3.8

x 1.1-1.9 mm, 1.4-2.2 mm body length perigynia, almost always smooth (0-1(16) teeth),

rounded to subacute base, beak often abruptly or subacutely contracted; beaks brown to

dark brown, apex orange or hyaline, mostly straight, teeth truncate, smooth, bifid;

gynoecium with 2 stigmas; rachilla often visible in relief on the side of abaxial

perigynia, setaceous, as long as or slightly surpassing; achenes ellipsoid, broadly

ellipsoid, lenticular or almost orbicular, the body grayish to light brown, non-glossy

surface, 1.4-2.6 x 1.0-1.9 mm, covering over 3/4 volume perigynia, at base broadly

truncated or rounded, apex obtuse or truncated; beaks absent or marked by the straight

base of the style.

Notes: C. cayouetteana subsp. altasierra can be differentiated by its short culms, as

long as the leaves; its staminate portion, as long as or slightly longer than the pistillate

portion, presenting a cone shape; its pistillate portion, densely packed. It occurs in high

91 ______Chapter 2. Taxonomy of the Carex capitata complex

elevations in California. Carex arctogena can be easily separated from C. cayouetteana

subsp. altasierra for having longer culms, longer spikes, straight tip leaves and for

having its lowermost perigynia horizontally orientated.

Distribution: Southwestern North American endemic (CAL).

Ecology: Non-glaciated plateaus and wet banks. 3400 - 3900 m.

92 ______Chapter 2. Taxonomy of the Carex capitata complex

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96 ______Chapter 2. Taxonomy of the Carex capitata complex

Appendix S1

97 ______Chapter 2. Taxonomy of the Carex capitata complex

Supporting Tables

Table 2.1: Diagnostic morphological characteristics used by Smith (1940) and Nilson (1991) to differentiate C. capitata from C. arctogena (taken from Reinhammar 1999).

Character Carex capitata Carex arctogena Spike size 6-9 mm long, light brownish- 3-6 mm long, dark brownish-green green Achene size On average 2.5 mm long and On average 1.9 mm long and 1.5 mm 1.8 mm wide wide Achene Pear-shaped, with a beak about More rounded, with a beak about 1/3 shape 1/5 of the total length; smooth of the total length; provided with 3-5 in the upper part small, sharp, teeth in the upper part

Pistillate Shorter than the achenes As long as the achenes scale length Beak length On average 0.4 mm On average 0.6 mm; achene more abruptly contracted into a beak Leaf length Leaves shorter than culms Leaves as long or longer than the culms Tussock Loose tussocks Dense tussocks density Habitat In rich mires, and along Wind-exposed heaths in rather dry demands riverlets; calciphilous; habitats; weakly calciphilous, also on lowlands subalpinelowalpine serpentine; mostly alpine, but occurs rarely in subalpine habitats

98 ______Chapter 2. Taxonomy of the Carex capitata complex

Table 2.2: Associates of C. arctogena in South America Locality Collection Elevation Associates No. (m) Argentina:Tierra J. Starr 60 Erigeron myosotis, Phleum alpinum, del Fuego, Rio 10015 & T. Caltha sagittata, Cerastium arvense, Grande Villaverde Carex macloviana and C. canescens

Argentina: Santa J. Starr 732 Nothofagus antartica, Cruz, Los 10020 & T. Marsippospermum grandiorum, Glaciares National Villaverde Chiliotrichum diffusum, Escallonia Park sp., Carex microglochin, C. banksii, C. atropicta, C. canescens,C. decidua, Gaultheria pumila, Empetrum rubrum and Rostkovia magellanica

Argentina: Santa J. Starr 449 Carex microglochin, C. magellanica, Cruz, Los 10023 & T. C. canescens, C. barrosii, Schoenus Glaciares National Villaverde andinus, Tetroncium magellanicum, Park Escallonia sp., Empetrum rubrum, Juncus sp., Rubus sp., Chiliotrichum diffusum, Blechnum penna-marina, Gaultheria pumila and Gavilea sp.

99 ______Chapter 2. Taxonomy of the Carex capitata complex

Table 2.3: Associates of C. cayouetteana subsp. cayouetteana. Locality Collection Elevation pH Associates No. (m) U.S.A.: Colorado, J. Starr 10S- 3602 5 Rhodiola sp., Castilleja sp., Lake Co., San 030, W. Potentilla sp., Salix spp.Bistorta Isabel National Sawtell & T. sp.,Caltha Forest Villaverde leptosepala,Pedicularis groenlandicum and Carex spp. U.S.A.: Colorado, J. Starr 10S- 3834 - Kobresia myosuroides Hinsdale Co., 033, W. Gunnison Sawtell & T. National Forest Villaverde U.S.A.: Montana, J. Starr 10S- 3137 5.4 Carex scirpoidea and Kobresia Carbon Co., 047A, W. myosuroides. It has also been Custer National Sawtell & T. reported to occur with Cassiope Forest, Absaroka- Villaverde mertensiana, Siebbaldia Beartooth procumbens and Stellaria spp. Wilderness U.S.A.: J. Starr 10S- 3291 - - Wyoming, Park 047B, W. Co., Shoshone Sawtell & T. National Forest, Villaverde Beartooth Plateau U.S.A.: J. Starr 10S- 1984 5 Phyllodoce empetriformis Washington, 061, W. Whatcom Co., Sawtell & T. Baker- Villaverde Snoqualmie National Forest

Table 2.4: Associates of C. cayouetteana subsp. bajasierra. Locality Collection Elevation pH Associates No. (m) U.S.A.: California, J. Starr 10S- 1441 7.7 Calocedrus decurrens, Butte Co., near 054 & T. Pseudotsuga menziesii, Cherry Hill Villaverde Pinus ponderosa, Abies Campground, Lassen magniffca, Darlingtonia National Forest californica, Drosera anglica, and Spiranthes sp. U.S.A.: Oregon, J. Starr 10S- 1927 5.8 Kobresia myosuroides Deschutes Co., 057 & T. Deschutes National Villaverde Forest

100 ______Chapter 2. Taxonomy of the Carex capitata complex

Table 2.5: Morphological characters studied. Continuous characters used by Egorova (1999) to differentiate between C. capitata and C. arctogena and those used in a pilot study to differentiate between C. cayouetteana subsp. cayouetteana, C. cayouetteana subsp. bajasierra and C. cayouetteana subsp. altasierra are denoted by asterisks. Character Definition Description Continuous variables CLMHT Culm length distance from the base of the culm to the base of the spike for the longest culm present (current and previous years)

CLMH* Culm length same as CLMHT but present year growth only CULMW Culm width width of the longest culm in the medial portion LEAFL Leaf length longest leaf from the base of the pseudoculm to the tip LEAFW Leaf width width of the longest leaf in the medial portion INFLOL* Inflorescence length maximum length from base of the spike to the bottom of the uppermost perigynium beak (=PERBKL) INFLOW* Inflorescence width maximum width of the spike from the base of the perigynium beak (=PERBKL)

MSPL* Inflorescence staminate length distance from the top of the portion proximal staminate scale to the apex FPPL* Inflorescence pistillate length distance from the base of the spike portion to the base of the most distance pistillate beak (=PERBKL) GLUMH* Length of the pistillate longest hyaline margin from the distal scale hyaline margin point of the proximal pistillate scale

GLUMHC* Length of the pistillate narrowest hyaline margin from the distal scale hyaline margin point of the proximal pistillate scale

FSCL* Length of the pistillate maximum scale length of the proximal scale perigynium FSCW* Pistillate scale width maximum scale width of the proximal perigynium FSCWL Maximum pistillate length distance from FSCW to the base of scale width the scale PERIGL* Perigynium length maximum length of the perigynium including the beak

101 ______Chapter 2. Taxonomy of the Carex capitata complex

PERBKL Beak length distance from distal point of the perigynium to the distal point of the achene PERIGW* Perigynium width maximum width of the perigynium PERIWD Maximum perigynium length distance from PERIGW to the base width of the perigynium ACHL Achene length maximum achene length ACHW Achene width maximum achene width MSCL* Staminate scale length maximum scale length at the medial part of the staminate portion of the inflorescence MSCW Staminate scale width maximum scale width at the medial point of the staminate portion of the inflorescence Discrete variables LEAFN Leaf number along the longest culm PSA Angle of the distal edge less than or greater than 45º of the pistillate scale

PERIGA Perigynium beak straight or bent inclination PERIGBo Perigynium angle less than or greater than 45º TEETHN Perigynium teeth along the margins of the perigynium number CULMD Culm teeth number number within the distal 1mm of the culm

Qualitative variable CULMC Culm sheath colour brown, red-brown, red-purple or purple- brown

102 ______Chapter 2. Taxonomy of the Carex capitata complex

Table 2.6: Mean ±1 SD and ranges for 22 morphological characters measured for the C. capitata complex. Character abbreviations correspond to those described in Table 2.5. All measurements are in millimeters. N = sample size.

Characte C. capitata C. arctogena C. C. C. r cayouetteana cayouetteana cayouetteana subsp. subsp. subsp. cayouetteana bajasierra altasierra (N=38) (N=35) (N=28) (N=24) (N=6) CLMHT 296.45±70.5 204.03±52.1 204.90±30.44 354.27±72.37 58.03±24.75 (150-490) (125.9-335) (142-260) (225-540) (140-205) CLMH 272.10±82.3 168.89±44.74 178.29±39.83 347.73±75.68 110.78±22.56 (120-490) (100-280) (116-260) (193-540) (83-140) LEAFL 205.92±50.0 154.64±47.9 157.49±21.11 206.97±41.68 113.08±19.27 (115-360) (90-298) (115-195) (133-270) (85-140) CULMW 0.75±0.09 0.78±0.15 0.89±0.12 0.88±0.08 0.83±0.12 (0.6-1) (0.5-1.1) (0.6-1.1) (0.7-1) (0.7-1) LEAFW 0.59±0.19 0.62±0.12 0.64±0.13 0.65±0.12 0.58±0.17 (0.4-1.5) (0.4-1) (0.4-0.9) (0.5-0.9) (0.4-0.9) INFLOW 4.41±0.48 3.83±0.39 4.44±0.61 3.86±0.43 3.07±0.69 (3.3-5.4) (2.9-4.7) (3.5-6.1) (3.2-4.6) (2.5-4) MSPL 1.95±0.65 2.12±0.68 3.22±1.40 5.36±2.00 3.05±1.03 (0.8-3.5) (1.2-3.7) (0.9-6.65) (2-10.5) (2.2-4.9) GLUMH 0.56±0.62 0.97±0.49 0.53±0.36 0.39±0.52 1.35±0.74 (0.01-2.25) (0.4-2.6) (0-1) (0.01-1.8) (0.01-2) GLUMH 0.19±0.15 0.40±0.17 0.23±0.16 0.09±0.10 0.27±0.18 C (0.01-0.5) (0.1-1) (0-0.5) (0.01-0.3) (0.01-0.5) INFLOL 7.52±1.20 7.34±1.16 9.11±1.63 11.25±2.33 7.65±0.87 (5.5-10.3) (5.2-9.8) (6.1-12.8) (6.8-16.9) (6.2-8.5) FPPL 4.78±0.89 4.46±0.64 5.00±0.80 5.08±0.83 3.70±0.84 (2.7-7.2) (3.5-6) (3.9-6.4) (3.9-6.9) (2-4.2) FSCL 2.12±0.25 2.18±0.29 2.43±0.24 2.18±0.33 1.93±0.12 (1.5-2.5) (1.4-3) (1.9-3) (1.2-2.7) (1.8-2.1) FSCW 1.43±0.21 1.77±0.34 1.73±0.27 1.74±0.24 1.53±0.35 (0.8-1.8) (1-2.6) (1.1-2.4) (1.4-2.2) (1.15-2) FSCWL 0.61±0.16 0.68±0.29 0.77±0.25 0.65±0.24 0.60±0.13 (0.3-1) (0.1-1.7) (0.1-1.3) (0.2-1) (0.4-0.8) PERIGL 2.99±0.45 2.65±0.45 2.80±0.38 2.35±0.45 2.55±0.64 (1.8-3.6) (1.5-3.2) (1.5-3.4) (1.5-3.1) (2-3.8) PERBKL 1.28±0.24 1.13±0.23 1.23±0.22 1.01±0.22 0.97±0.35 (0.8-1.7) (0.7-1.8) (0.9-1.9) (0.7-1.4) (0.6-1.6) PERIGW 1.79±0.21 1.50±0.19 1.96±0.31 1.66±0.17 1.50±0.26 (1.3-2.2) (1-2) (1.2-2.5) (1.2-2.2) (1.1-1.9) PERIWD 0.94±0.20 0.82±0.19 0.81±0.29 0.77±0.18 0.75±0.14 (0.5-1.3) (0.5-1.3) (0.3-1.8) (0.4-1) (0.5-0.9) ACHW 1.21±0.13 1.18±0.18 1.23±0.22 1.16±0.16 1.23±0.35

103 ______Chapter 2. Taxonomy of the Carex capitata complex

(1-1.5) (0.7-1.7) (0.6-1.8) (0.5-1.4) (1-1.9) ACHL 1.72±0.21 1.61±0.15 1.59±0.24 1.55±0.23 1.82±0.43 (1.1-2.1) (1.4-1.9) (1-2.3) (1-2) (1.4-2.6) MSCL 2.19±0.26 1.86±0.35 2.28±0.30 2.24±0.29 2.20±0.20 (1.6-2.9) (1-2.8) (1.8-3) (1.6-2.9) (1.9-2.5) MSCW 1.02±0.23 1.12±0.21 1.16±0.27 1.23±0.24 1.04±0.10 (0.6-1.5) (0.7-1.6) (0.6-1.9) (0.8-1.8) (0.9-1.2)

104 ______Chapter 2. Taxonomy of the Carex capitata complex

Table 2.7: Mean ± 1 SD and ranges for morphological characters measured for C. arctogena from South America vs. North America and Europe. Character abbreviations correspond to those described in Table 2.5. All measurements are in millimeters. N = sample size.

Character C. arctogena from C. arctogena from C. arctogena from Europe North America South America (N=10) (N=23) (N=6) CLMHT 179.52 ± 32.28 217.54 ± 58.46 221.28 ± 77.22 CULMW 7.70 ± 1.64 8.02 ± 1.47 8.00 ± 2.10 CLMH 153.10 ± 34.37 72.52 ± 50.73 195.99 ± 65.20 LEAFL 130.72 ± 29.53 60.61 ± 45.73 94.31 ± 70.21 LEAFW 6.00 ± 0.67 6.24 ± 1.35 6.83 ± 1.47 LEAFN 3.80 ± 0.42 3.52 ± 0.79 3.33 ± 0.52 INFLOW 36.30 ± 3.68 38.43 ± 3.82 43.17 ± 2.79 INFLOL 72.80 ± 13.85 71.48 ± 9.76 93.50 ± 20.54 MSPL 20.60 ± 7.76 20.04 ± 6.11 33.17 ± 13.09 FPPL 42.20 ± 6.71 44.96 ± 6.53 51.50 ± 5.47 GLUMH 8.90 ± 1.73 9.35 ± 5.36 12.33 ± 4.80 GLUMHC 4.40 ± 0.97 4.13 ± 1.98 4.00 ± 1.79 FSCL 22.00 ± 2.16 21.04 ± 2.48 24.67 ± 4.46 FSCW 17.40 ± 3.17 16.52 ± 3.10 22.67 ± 2.66 FSCWL 6.50 ± 2.59 7.26 ± 3.08 6.50 ± 3.39 PERIGL 25.20 ± 3.05 25.87 ± 4.98 30.50 ± 2.59 PERBKL 11.67 ± 3.24 10.95 ± 2.08 13.00 ± 1.55 PERIGW 14.70 ± 1.25 14.83 ± 2.04 16.83 ± 2.48 PERIWD 6.80 ± 1.69 8.91 ± 2.56 9.00 ± 3.16 TEETHN 4.56 ± 1.13 4.22 ± 3.15 7.83 ± 3.87 ACHW 11.89 ± 1.54 11.41 ± 2.02 12.67 ± 1.51 ACHL 15.78 ± 1.56 16.36 ± 1.36 15.83 ± 1.72 MSCL 18.50 ± 3.69 18.22 ± 2.52 23.67 ± 5.68 MSCW 11.20 ± 2.66 10.89 ± 1.35 12.83 ± 3.19 CULMD 5.70 ± 3.13 8.30 ± 4.34 7.50 ± 5.01

Table 2.8: Mann-Whitney significance for pairwise comparisons of each signifcant variable from the Kruskal-Wallis test, ordered by its utility to significantly differentiate between taxa. N denotes no significance, Y denotes significance with p<0.05 and Y* denotes significance with p <0.01. Variables in bold were included in the Principal components analysis. Carex capitata (C), C. arctogena (A), C. cayouetteana subsp. cayouetteana (Y), C. cayouetteana subsp. bajasierra (Y2) and C. cayouetteana subsp. altasierra (Y3).

105 ______Chapter 2. Taxonomy of the Carex capitata complex

Variable Y3-Y2 Y3-Y Y3-A Y3-C Y2-Y Y2-A Y2-C Y-A Y-C A-C CLMH Y Y Y Y Y* Y* Y N Y* Y* CLMHT Y N Y Y Y* Y* Y N Y* Y* MSPL N Y Y Y Y Y* Y* Y Y N LEAFL Y Y N Y Y* Y* N N Y* Y* INFLOL Y N N N Y Y* Y* Y* Y N PERIGW N N N N Y Y Y Y* Y Y* GLUMHC N N N N Y Y* Y Y N Y* INFLOW N Y N N Y* N Y* Y* N Y* FSCL N Y N N Y N N Y* Y* N MSCW N N N N N Y Y N Y* Y FSCW N N N N N N Y* N Y* Y* MSCL N N N N N Y* N Y* N Y CULMW N N N N N Y* Y Y* N N GLUMH N N N N N Y* N Y N Y ACHL N N N N N N Y N Y Y FPPL Y N N N N Y N Y N N PERIBKL N N N N Y N Y N N Y PERIGL N N N N Y N Y N N Y FSCWL N N N N N N N N Y N PERIWD N N N N N N Y N N N

106 ______Chapter 2. Taxonomy of the Carex capitata complex

Table 2.9: Percentage of the total variance explained by principal component scores of the variables included in di erent PCAs. PC = Ordered principal Component. PC All 12 variables variables 1 19,401 28,53 2 12,4 16,709 3 10,989 12,48 4 7,6767 10,307 5 6,7801 7,8787 6 6,0676 6,4935 7 5,2372 5,0133 8 4,3777 4,467 9 3,8413 3,0117 10 3,4106 2,774 11 3,0812 2,1061 12 2,899 0,23034 13 2,7171 14 2,2072 15 1,9515 16 1,7454 17 1,3808 18 1,347 19 1,1537 20 1,0073 21 0,21384 22 0,1153 Total 100 100

107 ______Chapter 2. Taxonomy of the Carex capitata complex

Supporting Figures

Figure 1. The distribution of C. arctogena based on all the herbarium specimens

examined in this study. Inset represents the distribution in Scandinavia.

108 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 2. Distribution of C. capitata, C. arctogena, C. cayouetteana subsp.

cayouetteana, C. cayouetteana subsp. bajasierra and C. cayouetteana subsp. altasierra

herbarium specimens used in the morphological study.

109 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 3. Photographs of herbarium sheets of C. cayouetteana subsp. bajasierra

identified as C. capitata from CHSC. Inset shows spike and perigynium details.

110 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 4. Photographs of herbarium sheets of C. cayouetteana subsp. altasierra

identified as C. arctogena from CAL. Inset shows spike and perigynium details.

111 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 5. Photographs of herbarium sheets of C. cayouetteana subsp. cayouetteana

identified as C. arctogena from COLO. Inset shows spike and perigynium details.

112 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 6. Photographs of herbarium sheets of C. arctogena form H. Inset shows spike

and perigynium details.

113 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 7. Photographs of a herbarium sheet of C. capitata from H. Inset shows spike

and perigynium details.

114 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 8. PCA scatter plot of the first two principal component using C. arctogena

specimens from Europe (circles), North America (triangles) and South America

(crosses) and 12 quantitative variables.

115 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 9. PCA scatter plot of the first two components using all C. arctogena and C.

capitata specimens studied and 12 quantitative variables. Symbols represent C. capitata

(circles), C. arctogena from the Northern Hemisphere (triangles) and C. arctogena from

the Southern Hemisphere (crosses).

116 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 10. PCA scatter plot of the first two components using all specimens studied of

C. arctogena (crosses), C. cayouetteana subsp. cayouetteana (dark gray traingles), C.

cayouetteana subsp. bajasierra (medium gray triangles) and C. cayouetteana subsp.

altasierra (light gray trinalges) and 12 quantitative variables.

Figure 11. PCA scatter plot of the first two components using all specimens studied of

117 ______Chapter 2. Taxonomy of the Carex capitata complex

C. cayouetteana subsp. cayouetteana (dark gray traingles), C. cayouetteana subsp.

bajasierra (medium gray triangles) and C. cayouetteana subsp. altasierra (light gray

trinalges) and 12 quantitative variables.

118 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 12. PCA scatter plot of the first two components using all specimens of C.

arctogena (crosses), C. cayouetteana subsp. cayouetteana (medium gray triangles) and

C. cayouetteana subsp. altasierra (light gray triangles) and 12 quantitative variables.

119 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 13. PCA scatter plot of the first two components using C. arctogena (crosses)

and C. cayouetteana subsp. altasierra (light gray triangles) specimens and 12

quantitative variables.

120 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 14. Scanning electron photographs of silica bodies of all putative taxa in the C.

capitata complex. (A) Carex capitata, A. Dutilly & E. Lepage 16761 (CAN-17332)

from Ontario; (B) C. arctogena, J. Starr 10023 & T. Villaverde (CAN) from Argentina;

(C) C. cayouetteana subsp. cayouetteana, K. H. Lackschewitz 9909 (MONTU-86558)

from Montana;(D) C. cayouetteana subsp. bajasierra, J. Starr & J. Thibeault 07-44

from California (CAN); (E) C. cayouetteana subsp. altasierra, C. W. Sharsmith 2681

(CAN-162869). See Table A.9 for additional specimen voucher information.

121 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure 15. The distribution of C. capitata based on all the herbarium specimens

examined in this study. Inset represents the distribution in Scandinavia.

122 ______Chapter 2. Taxonomy of the Carex capitata complex

Additional information

123 ______Chapter 2. Taxonomy of the Carex capitata complex

Table A.1: Kruskal-Wallis test. Chi-square value, degrees of freedom (df) and P-value are shown for each variable. Variable Kruskal- chi-square p- value Wallis df CLMHT 76.238 4 1,09E-12 CLMH 780.187 4 4,58E-13 LEAFL 447.911 4 4,39E-06 CULMW 299.697 4 4,96E-03 LEAFW 69.523 4 0.1384 INFLOW 442.021 4 5,83E-06 MSPL 592.884 4 4,09E-09 GLUMH 28.639 4 9,26E-03 GLUMHC 451.364 4 3,73E-06 INFLOL 549.027 4 3,41E-08 FPPL 179.482 4 0.001263 FSCL 28.635 4 9,27E-03 FSCW 334.419 4 9,70E-04 FSCWL 104.719 4 0.03319 PERIGL 321.547 4 1,78E-03 PERBKL 237.847 4 8,82E-02 PERIGW 532.796 4 7,45E-08 PERIWD 132.667 4 0.01004 ACHW 31.064 4 0.5402 ACHL 145.449 4 0.005745 MSCL 307.368 4 3,46E-03 MSCW 127.065 4 0.01280

124 ______Chapter 2. Taxonom______y of the Carex capitata complex

Table A.2: Correlation matrix for 22 continuous variables used in the morphometric study. LEAFW MSCL CLMH CLMHT LEAFL CULMW INFLO W MSPL GLUMH GLUMH C INFLOL FPPL FSCL FSCW FSCWL PERIGL PERBKL PERIG W PERIWD ACHW ACHL MSCW CLMHT 1,0 CLMH 0,9 1,0 LEAFL 0,8 0,7 1,0 CULMW 0,0 0,0 0,1 1,0 LEAFW 0,0 0,0 0,1 0,2 1,0 INFLOW 0,1 0,1 0,2 0,1 0,1 1,0 MSPL 0,3 0,4 0,2 0,3 0,1 0,0 1,0 GLUMH 0,2 0,2 0,1 0,2 0,1 0,1 0,1 1,0 GLUMHC 0,3 0,3 0,2 0,1 0,0 0,1 0,2 0,6 1,0 INFLOL 0,4 0,4 0,3 0,3 0,1 0,2 0,9 0,1 0,2 1,0 FPPL 0,2 0,2 0,2 0,2 0,1 0,5 0,2 0,2 0,1 0,5 1,0 FSCL 0,0 0,0 0,1 0,3 0,1 0,3 0,2 0,0 0,1 0,3 0,2 1,0 FSCW 0,1 0,0 0,0 0,2 0,1 0,1 0,3 0,2 0,1 0,3 0,2 0,4 1,0 FSCWL 0,0 0,0 0,0 0,2 0,0 0,0 0,2 0,0 0,1 0,2 0,0 0,4 0,2 1,0 PERIGL 0,0 0,1 0,1 0,2 0,2 0,4 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,2 1,0 PERBKL 0,0 0,0 0,1 0,0 0,0 0,5 0,1 0,0 0,0 0,1 0,2 0,2 0,1 0,0 0,5 1,0 PERIGW 0,1 0,1 0,1 0,2 0,1 0,5 0,1 0,2 0,2 0,2 0,2 0,3 0,0 0,2 0,4 0,3 1,0 PERIWD 0,0 0,1 0,0 0,1 0,1 0,1 0,2 0,1 0,0 0,2 0,1 0,1 0,1 0,1 0,2 0,1 0,0 1,0 ACHW 0,1 0,0 0,1 0,1 0,1 0,2 0,0 0,1 0,1 0,0 0,0 0,1 0,0 0,1 0,3 0,2 0,3 0,1 1,0 ACHL 0,0 0,0 0,0 0,0 0,1 0,1 0,0 0,1 0,0 0,0 0,0 0,0 0,1 0,1 0,2 0,1 0,1 0,0 0,5 1,0 MSCL 0,2 0,2 0,2 0,1 0,1 0,4 0,3 0,1 0,2 0,4 0,3 0,2 0,1 0,0 0,2 0,1 0,4 0,0 0,2 0,1 1,0 MSCW 0,1 0,1 0,1 0,2 0,1 0,0 0,4 0,1 0,0 0,4 0,0 0,2 0,3 0,2 0,1 0,1 0,1 0,1 0,1 0,0 0,2 1

125 ______Chapter 2. Taxonomy of the Carex capitata complex

Table A.4: Summary statistics for the morphometric analysis of C. arctogena. Abbreviations: n = sample size; sd = standard deviation; mad = median absolute deviation; se= standard error. Variable n mean sd median trimmed mad min max range skew kurtosis se CLMHT 35 204.03 52.16 190.5 200.03 45.29 125.9 335.05 209.15 0.68 -0.29 8.82 CLMH 35 168.89 44.74 169.0 165.20 38.62 100.7 280.05 179.35 0.64 -0.09 7.56 LEAFL 35 154.64 47.90 145.5 150.61 52.34 90.5 298.70 208.20 0.87 0.32 8.10 CULMW 35 0.78 0.15 0.8 0.78 0.15 0.5 1.10 0.60 0.03 -0.97 0.03 LEAFW 35 0.62 0.12 0.6 0.61 0.15 0.4 1.00 0.60 0.65 0.98 0.02 INFLOW 35 3.83 0.39 3.8 3.83 0.44 2.9 4.70 1.80 -0.02 -0.46 0.07 MSPL 35 2.12 0.68 2.0 2.09 0.74 1.2 3.70 2.50 0.46 -0.95 0.12 GLUMH 35 0.97 0.49 0.9 0.88 0.30 0.4 2.60 2.20 1.70 2.56 0.08 GLUMHC 35 0.40 0.17 0.4 0.40 0.15 0.1 1.00 0.90 0.98 2.64 0.03 INFLOL 35 7.34 1.16 7.4 7.30 1.19 5.2 9.80 4.60 0.41 -0.64 0.20 FPPL 35 4.46 0.64 4.5 4.43 0.74 3.5 6.00 2.50 0.34 -0.51 0.11 FSCL 35 2.18 0.29 2.1 2.17 0.15 1.4 3.00 1.60 0.43 1.15 0.05 FSCW 35 1.77 0.34 1.8 1.77 0.30 1.0 2.60 1.60 0.11 0.04 0.06 FSCWL 35 0.68 0.29 0.6 0.66 0.15 0.1 1.70 1.60 1.22 2.79 0.05 PERIGL 35 2.65 0.45 2.7 2.72 0.44 1.5 3.20 1.70 -1.19 0.81 0.08 PERBKL 35 1.13 0.23 1.2 1.12 0.30 0.7 1.80 1.10 0.50 0.80 0.04 PERIGW 35 1.50 0.19 1.5 1.50 0.15 1.0 2.00 1.00 -0.03 0.58 0.03 PERIWD 35 0.82 0.19 0.8 0.82 0.15 0.5 1.30 0.80 0.26 -0.41 0.03 ACHW 35 1.18 0.18 1.2 1.18 0.15 0.7 1.70 1.00 0.07 1.34 0.03 ACHL 35 1.61 0.15 1.6 1.60 0.15 1.4 1.90 0.50 0.61 -0.55 0.03 MSCL 35 1.86 0.35 1.8 1.86 0.30 1.0 2.80 1.80 0.17 0.41 0.06 MSCW 35 1.12 0.21 1.1 1.11 0.15 0.7 1.60 0.90 0.40 -0.36 0.04

126 ______Chapter 2. Taxonomy of the Carex capitata complex

Table A.5: Summary statistics for the morphometric analysis of C. capitata. Abbreviations: n = sample size; sd = standard deviation; mad = median absolute deviation; se= standard error. Variable n mean sd median trimmed mad min max range skew kurtosis se CLMHT 38 296.45 70.50 290.05 293.55 70.42 150.05 490.05 340.00 0.49 0.12 11.44 CLMH 38 272.10 82.37 270.70 268.12 66.72 120.05 490.05 370.00 0.53 -0.14 13.36 LEAFL 38 205.92 50.00 210.38 203.87 43.70 115.05 360.05 245.00 0.64 0.70 8.11 CULMW 38 0.75 0.09 0.70 0.74 0.15 0.60 1.00 0.40 0.45 0.46 0.01 LEAFW 38 0.59 0.19 0.60 0.57 0.15 0.40 1.50 1.10 2.78 10.06 0.03 INFLOW 38 4.41 0.48 4.45 4.43 0.52 3.30 5.40 2.10 -0.14 -0.50 0.08 MSPL 38 1.95 0.65 1.85 1.93 0.74 0.80 3.50 2.70 0.25 -0.77 0.11 GLUMH 38 0.56 0.62 0.50 0.46 0.59 0.01 2.25 2.24 1.39 1.24 0.10 GLUMHC 38 0.19 0.15 0.15 0.18 0.21 0.01 0.50 0.49 0.21 -1.44 0.02 INFLOL 38 7.52 1.20 7.40 7.51 1.33 5.50 10.30 4.80 0.16 -0.90 0.19 FPPL 38 4.78 0.89 4.60 4.71 0.67 2.70 7.20 4.50 0.74 0.92 0.14 FSCL 38 2.12 0.25 2.20 2.14 0.22 1.50 2.50 1.00 -0.72 0.03 0.04 FSCW 38 1.43 0.21 1.40 1.43 0.15 0.80 1.80 1.00 -0.49 0.60 0.03 FSCWL 38 0.61 0.16 0.60 0.60 0.15 0.30 1.00 0.70 0.16 -0.28 0.03 PERIGL 38 2.99 0.45 3.10 3.04 0.30 1.80 3.60 1.80 -1.09 0.69 0.07 PERBKL 38 1.28 0.24 1.30 1.29 0.30 0.80 1.70 0.90 -0.39 -0.74 0.04 PERIGW 38 1.79 0.21 1.80 1.79 0.30 1.30 2.20 0.90 -0.30 -0.52 0.03 PERIWD 38 0.94 0.20 1.00 0.94 0.22 0.50 1.30 0.80 -0.26 -0.86 0.03 ACHW 38 1.21 0.13 1.20 1.21 0.15 1.00 1.50 0.50 0.08 -0.69 0.02 ACHL 38 1.72 0.21 1.70 1.73 0.15 1.10 2.10 1.00 -0.86 0.82 0.03 MSCL 38 2.19 0.26 2.20 2.19 0.15 1.60 2.90 1.30 0.17 0.46 0.04 MSCW 38 1.02 0.23 1.00 1.02 0.30 0.60 1.50 0.90 0.16 -0.81 0.04

127 ______Chapter 2. Taxonomy of the Carex capitata complex

Table A.6: Summary statistics for the morphometric analysis of Carex cayouetteana subsp. cayouetteana. Abbreviations: n = sample size; sd = standard deviation; mad = median absolute deviation; se= standard error. Variable n mean sd median trimmed mad min max range skew kurtosis se CLMHT 28 204.90 30.44 205.23 205.15 22.24 142.72 260.50 117.78 -0.09 -0.25 5.75 CLMH 28 178.29 39.83 182.12 177.94 51.32 116.00 260.05 144.05 -0.04 -1.16 7.53 LEAFL 28 157.49 21.11 160.05 157.93 25.39 115.05 195.70 80.65 -0.15 -1.00 3.99 CULMW 28 0.89 0.12 0.90 0.89 0.15 0.60 1.10 0.50 -0.33 -0.34 0.02 LEAFW 28 0.64 0.13 0.60 0.63 0.15 0.40 0.90 0.50 0.29 -0.62 0.02 INFLOW 28 4.44 0.61 4.35 4.39 0.52 3.50 6.10 2.60 1.07 0.61 0.11 MSPL 28 3.22 1.40 3.25 3.13 1.11 0.90 6.65 5.75 0.50 0.05 0.27 GLUMH 28 0.53 0.36 0.50 0.53 0.52 0.00 1.00 1.00 -0.08 -1.43 0.07 GLUMHC 28 0.23 0.16 0.25 0.23 0.22 0.00 0.50 0.50 0.07 -1.27 0.03 INFLOL 28 9.11 1.63 9.40 9.09 1.56 6.10 12.80 6.70 0.04 -0.63 0.31 FPPL 28 5.00 0.80 5.00 4.97 0.96 3.90 6.40 2.50 0.23 -1.34 0.15 FSCL 28 2.43 0.24 2.45 2.42 0.22 1.90 3.00 1.10 0.09 0.27 0.05 FSCW 28 1.73 0.27 1.65 1.72 0.22 1.10 2.42 1.32 0.27 0.09 0.05 FSCWL 28 0.77 0.25 0.77 0.78 0.22 0.10 1.30 1.20 -0.17 0.38 0.05 PERIGL 28 2.80 0.38 2.90 2.84 0.30 1.50 3.40 1.90 -1.45 2.98 0.07 PERBKL 28 1.23 0.22 1.20 1.20 0.15 0.90 1.90 1.00 1.44 1.95 0.04 PERIGW 28 1.96 0.31 1.90 1.99 0.30 1.20 2.50 1.30 -0.59 0.34 0.06 PERIWD 28 0.81 0.29 0.75 0.80 0.37 0.30 1.80 1.50 1.13 2.59 0.05 ACHW 28 1.23 0.22 1.20 1.22 0.15 0.60 1.80 1.20 0.16 2.34 0.04 ACHL 28 1.59 0.24 1.60 1.59 0.15 1.00 2.30 1.30 0.32 1.38 0.05 MSCL 28 2.28 0.30 2.20 2.26 0.30 1.80 3.00 1.20 0.47 -0.31 0.06 MSCW 28 1.16 0.27 1.13 1.16 0.19 0.60 1.90 1.30 0.28 0.75 0.05

128 ______Chapter 2. Taxonomy of the Carex capitata complex

Table A.7: Summary statistics for the morphometric analysis of C. cayouetteana subsp. bajasierra. Abbreviations: n = sample size; sd = standard deviation; mad = median absolute deviation; se= standard error.

Variable n mean sd median trimmed mad min max range skew kurtosis se CLMHT 28 204.90 30.44 205.23 205.15 22.24 142.72 260.50 117.78 -0.09 -0.25 5.75 CLMH 28 178.29 39.83 182.12 177.94 51.32 116.00 260.05 144.05 -0.04 -1.16 7.53 LEAFL 28 157.49 21.11 160.05 157.93 25.39 115.05 195.70 80.65 -0.15 -1.00 3.99 CULMW 28 0.89 0.12 0.90 0.89 0.15 0.60 1.10 0.50 -0.33 -0.34 0.02 LEAFW 28 0.64 0.13 0.60 0.63 0.15 0.40 0.90 0.50 0.29 -0.62 0.02 INFLOW 28 4.44 0.61 4.35 4.39 0.52 3.50 6.10 2.60 1.07 0.61 0.11 MSPL 28 3.22 1.40 3.25 3.13 1.11 0.90 6.65 5.75 0.50 0.05 0.27 GLUMH 28 0.53 0.36 0.50 0.53 0.52 0.00 1.00 1.00 -0.08 -1.43 0.07 GLUMHC 28 0.23 0.16 0.25 0.23 0.22 0.00 0.50 0.50 0.07 -1.27 0.03 INFLOL 28 9.11 1.63 9.40 9.09 1.56 6.10 12.80 6.70 0.04 -0.63 0.31 FPPL 28 5.00 0.80 5.00 4.97 0.96 3.90 6.40 2.50 0.23 -1.34 0.15 FSCL 28 2.43 0.24 2.45 2.42 0.22 1.90 3.00 1.10 0.09 0.27 0.05 FSCW 28 1.73 0.27 1.65 1.72 0.22 1.10 2.42 1.32 0.27 0.09 0.05 FSCWL 28 0.77 0.25 0.77 0.78 0.22 0.10 1.30 1.20 -0.17 0.38 0.05 PERIGL 28 2.80 0.38 2.90 2.84 0.30 1.50 3.40 1.90 -1.45 2.98 0.07 PERBKL 28 1.23 0.22 1.20 1.20 0.15 0.90 1.90 1.00 1.44 1.95 0.04 PERIGW 28 1.96 0.31 1.90 1.99 0.30 1.20 2.50 1.30 -0.59 0.34 0.06 PERIWD 28 0.81 0.29 0.75 0.80 0.37 0.30 1.80 1.50 1.13 2.59 0.05 ACHW 28 1.23 0.22 1.20 1.22 0.15 0.60 1.80 1.20 0.16 2.34 0.04 ACHL 28 1.59 0.24 1.60 1.59 0.15 1.00 2.30 1.30 0.32 1.38 0.05 MSCL 28 2.28 0.30 2.20 2.26 0.30 1.80 3.00 1.20 0.47 -0.31 0.06 MSCW 28 1.16 0.27 1.13 1.16 0.19 0.60 1.90 1.30 0.28 0.75 0.05

129 ______Chapter 2. Taxonomy of the Carex capitata complex

Table A.8: Summary statistics for the morphometric analysis of C. cayouetteana subsp. altasierra. Abbreviations: n = sample size; sd = standard deviation; mad = median absolute deviation; se= standard error.

Variable n mean sd median trimmed mad min max range skew kurtosis se CLMHT 6 158.03 24.75 147.53 158.03 9.25 140.50 205.05 64.55 1.01 -0.74 10.10 CLMH 6 110.78 22.56 112.53 110.78 26.39 83.97 140.05 56.08 0.03 -2.01 9.21 LEAFL 6 113.08 19.27 114.03 113.08 18.35 85.00 140.05 55.05 -0.07 -1.55 7.87 CULMW 6 0.83 0.12 0.85 0.83 0.15 0.70 1.00 0.30 0.04 -1.88 0.05 LEAFW 6 0.58 0.17 0.55 0.58 0.07 0.40 0.90 0.50 0.80 -0.86 0.07 INFLOW 6 3.07 0.69 2.70 3.07 0.22 2.50 4.00 1.50 0.51 -1.94 0.28 MSPL 6 3.05 1.03 2.85 3.05 0.82 2.20 4.90 2.70 0.77 -1.06 0.42 GLUMH 6 1.35 0.74 1.45 1.35 0.59 0.01 2.00 1.99 -0.77 -0.99 0.30 GLUMHC 6 0.27 0.18 0.30 0.27 0.22 0.01 0.50 0.49 -0.20 -1.76 0.07 INFLOL 6 7.65 0.87 8.00 7.65 0.52 6.20 8.50 2.30 -0.62 -1.48 0.36 FPPL 6 3.70 0.84 4.00 3.70 0.00 2.00 4.20 2.20 -1.33 -0.13 0.34 FSCL 6 1.93 0.12 1.95 1.93 0.15 1.80 2.10 0.30 0.04 -1.88 0.05 FSCW 6 1.53 0.35 1.46 1.53 0.43 1.15 2.00 0.85 0.23 -1.94 0.14 FSCWL 6 0.60 0.13 0.60 0.60 0.00 0.40 0.80 0.40 0.00 -0.92 0.05 PERIGL 6 2.55 0.64 2.40 2.55 0.22 2.00 3.80 1.80 1.09 -0.48 0.26 PERBKL 6 0.97 0.35 0.95 0.97 0.22 0.60 1.60 1.00 0.72 -0.96 0.14 PERIGW 6 1.50 0.26 1.50 1.50 0.15 1.10 1.90 0.80 0.00 -1.15 0.11 PERIWD 6 0.75 0.14 0.80 0.75 0.07 0.50 0.90 0.40 -0.76 -0.95 0.06 ACHW 6 1.23 0.35 1.10 1.23 0.15 1.00 1.90 0.90 1.05 -0.63 0.14 ACHL 6 1.82 0.43 1.75 1.82 0.30 1.40 2.60 1.20 0.81 -0.89 0.17 MSCL 6 2.20 0.20 2.20 2.20 0.15 1.90 2.50 0.60 0.00 -1.29 0.08 MSCW 6 1.04 0.10 1.04 1.04 0.07 0.90 1.20 0.30 0.10 -1.45 0.04

130 ______Chapter 2. Taxonomy of the Carex capitata complex

Studied specimens of C. arctogena Argentina, Chubut, Los Alerces National Park, Soriano, A., 30.3.1952, (BAA). Dept. Chos Malal, 2300 m, Boelcke, O., Correa, M.N.; Bacigalupo, N.M., 30.1.1964, (BAA, 11368). Mendoza, Cordillera del Rio Barrancas, Kurtz, F., 16.11.1888, (MICH). Canada, Alberta, Mercoal, Rousseau, J., 18.7.1947, (COLO, 13811). Alberta, Mercoal, 4300 ft, Malte, M.O., Watson, W.R., 8.8.1925, (RM, 280606). British Columbia, Pine Pass, 1402 m, Argus, G.W., 12.7.1973, (CAN, 372267). British Columbia, 7228 ft, Calder, J., 149035, Parmelee, J.A.; Taylor, R.L., 8.8.1956, (COLO, 149035). British Columbia, Mount Apex, 7100 ft, Calder, J., Savile, O., 11.8.1953, (RM, 252249). Manitoba, Fort Chimo, Rousseau, J., 14.8.1951, (WIN, 22355). Manitoba, Baralzon Lake, Scoggan, H.J., 22434, Baldwin, W.K.W., 28.7.1950, (WIN, 22434). Manitoba, Hudsons Bay Co., Duck Lake, Scoggan, H.J., Baldwin, W.K.W., 10.8.1950, (WIN, 22435). Manitoba, Fort Chimo, Legault, A., 22.7.1963, (COLO, 491481). Manitoba, Hudsons Bay Co., Duck Lake, Scoggan, H.J., Baldwin, W.K.W., 10.8.1950, (CAN, 201506). Manitoba, Baralzon Lake, Scoggan, H.J., Baldwin, W.K.W., 30.7.1950, (CAN, 202500). Manitoba, Nueltin Lake, Baldwin, W.K.W., 26.7.1951, (CAN, 212816). Manitoba, Cochrane River, Baldwin, W.K.W., 3.7.1951, (CAN, 212817). Manitoba, Cochrane River, Baldwin, W.K.W., 3.7.1951, (CAN, 212817). Manitoba, Baralzon Lake, Scoggan, H.J.,Baldwin, W.K.W., 28.7.1950, (CAN, 201507). Newfoundland-Labrador, Esker area, Mäkinen, Y., Kankainen, E. 21.7.1967, (CAN, 314758). Newfoundland-Labrador, Esker area, 838 m, Mäkinen, Y.Kankainen, E.21.7.1967, (CAN, 314758). Newfoundland-Labrador, Twin Falls, Hustich, I., 6.7.1967, (CAN, 313311). Nunavut, Upper Hood River, Gould, W., 7.1995, (COLO, 475773). Ontario, Kenora District, Patricia PortionRiley, J.L., 12.8.1980, (CAN). Ontario, Hudson Bay Lowlands, Porsild, A.E., Baldwin, W.K.W.4.7.1957, (CAN, 278707). Quebec, Fort Chimo, Sørensen, T.H., 17.8.1959, (C). Quebec, Baie dUngava, Blondeau, M., 1.8.1993, (WIN, 53902). Quebec, Baie dUngava, Rousseau, J., 23.7.1951, (WIN, 22356). Quebec,Lac Jaucourt Region Lichteneger Lake,487 m Argus, G.W., 16.7.1974, (CAN, 3779977). Quebec, Boatswain Bay, Baldwin, W.K.W., 17333, Hustich, I.; Kucyniak, J.; Tuomikoski, R., 8.7.1947, (CAN, 17333). Quebec, Lac Payne, Legault, A., 23398, 2.8.1965, (CCO, 23398). Quebec, Northern QuebecLake Payne, Legault, A.,Brisson, S. 2.8.1965, (COLO, 210789). Quebec, Ungava, Husons Bay, Dutilly, A., Lepage, E., 21.3.1945, (RM, 233644). Quebec, Fort Chimo, Calder, J., 31.7.1948, (RM, 255325). Quebec,Hudson Bay Cairn Island, Abbe, E.C.,Abbe, L.B.; Marr, J. 30.7.1939, (RM, 252521). Quebec, Hudson Bay,Great Whale River Calder, J.Savile, O.; Kukkonen, I., 8.8.1959, (RM, 260486). Quebec, Lac Kopeteokash, Rousseau, J., 18.7.1947, (RM, 228636). Saskatchewan, Vicinity of Patterson Lake, Argus, G.W., 20.7.1963, (CAN, 282691). Saskatchewan, Vicinity of Patterson Lake, Argus, G.W., 20.7.1963, (CAN, 282691).Saskatchewan, Northeastern SaskatchewanPatterson Lake , Argus, G.W., 20.7.1963, (RM, 277437). Enontekiö, KilpisjärviSaana, 750 m Roivainen, L., 8.7.1935, (H, 127310). Enontekiö, KilpisjärviSaana, 750 mVäre, H., 29.7.2004, (H, 805587). Enontekiö Lapland, 825 m, Väre, H., 17.7.2006, (H, 809948). Inari, Vätsäri Wilderness Area, Kulmala, H., 27.7.1996, (H, 717201). Lapponia Imandrae, Lindén, J., 18.7.1891, (H, 325665). Lapponia Imandrae, Axelson, W.M., Borg, V., 24.7.1901, (H, 325667).Finland, Lapponia murmanica, 550 m, Brotherus, V.F., 8.1887, (H, 325639). PetsamoCajander, A., 10.7.1927, (H, 325644). Porojärvet, Toskalhar950 m,Roivainen, H.Ollila, L. 15.7.1955, (H, 127313). Porojärvet, Toskalhar, 910 m, Roivainen, H., 15.7.1966, (H, 179889). Foutell, C.W., Jalan, M.J., 10.8.1899, (H, 325657). Altevatn, 500 m, 17.8.1967, (M, 0151943). Groenlandia meridionalis, Kangerdluarssuk, Hansen, C. 282521,Hansen, K.; Petersen, M. 4.7.1962, (CAN).Nigerdleq, Jørgensen, L.B. 15.7.1966, (CAN, 311369). Greenland, Vestgrønland, Pingorssuaq Kitdleq, 400 mHanfgarn, S., 11.8.1983, (C). Tugtilik Lake, 10 m, Elsley, J.E. 15.8.1967, (M, 0151948). Lagerkranz, J., 2.8.1936, (RMS, 153944). Finnmark,Sör-Varanger Bugöynes, Toivonen, H., 30.7.1971, (H, 1081734). Finnmark, Sör-Varanger, Bugöynes, Toivonen, H., 1081733, 30.7.1971, (H, 1081733). Nordland, Narvik hd., Skjomen, Skifte, O., GRaff, G.; Spjelkavik, S., 11.8.1973 (H). Norland, Sulitjelma, Skifte, O. 1.8.1962, (DAO, 285800). Sverige, Abisko, Paddas, Lid, J., 2.8.1950, (H, 1300264). Norway, Troms, Bardu, Leinavatn, 498 mEngelskjøn, T.,Engelskjøn, E.M. 7.7.1977, (C). Troms, Bardu, Altevatn580 m, 18.8.1967, (M, 0151942). Troms, Bardu,Kampaksla 780 m, Engelskjøn, T.,Skifte, O. 9.8.1978, (H, 1685049). Petsamo, Petchenga Vouvatusjärvi, Piirainen, M., 27.7.1995, (H, 1682990). Sweden,Torne

131 ______Chapter 2. Taxonomy of the Carex capitata complex

Lappmark, Karesuando, 1000 m, Smith, H., 26.7.1933, (DAO, 257429). Torne Lappmark, Karesuando, 1000 m, Smith, H., 26.7.1993, (H, 1652844). Torne Lappmark, Jukkasjärvi parish, 550 m, Alm, G., Smith, H. 23.7.1939, (H, 1300259). New Hampshire, Coos Co., Mt. Washington, Hodgon, A.R., Gale, M., 30.6.1950, (DAO, 257427). New Hampshire, White Mountains, Mt. Washington, Forbes, F., 9.8.1902, (RMS, 242089). New Hampshire, Alpine Garden, Mt. Washington, Sargent, F.H., 5.7.1942, (BRY, 143916). New Hampshire, Alpine Garden, Mt. Washington,5000 ft Löve, A.,Löve, D. 27.7.1958, (COLO, 288736). New Hampshire, Alpine Garden, Mt. Washington, Löve, A. ,Löve, D. 3.7.1960, (COLO, 295019). New Hampshire, White MountainsMt. Washington, Forbes, F., 9.8.1902, (RM, 50212).

Studied specimens of C. capitata Austria, Innsbruck, Seefeld, 1180 m, HöllerJ. s.n., 26.7.1958, (M, 0151923). Tirol, Seiser Alp, 2000 m, Görz s.n., 27.7.1914, (GH). Canada, Alberta, Ft. Fitzgerald, Cody, W.J. 4533 and Loan, C.C., 19.7.1950, (RM, 228683). British Columbia, Bluster Mt., 2133 m, Thompson, J. s.n. and Thompson, M., 14.7.1938, (WTU, 17326). British Columbia, Mt. Tinsdale, 2133 m, Krajina, J. s.n. and Pojar, J., 13.8.1974, (UBC, 149191). British Columbia, Mount Apex, 2164 m, Calder, J. 11795 and Savile, O., 11.8.1953, (WTU, 170234). British Columbia, Anahim Lake, 1219 m, Calder, J. 18578, Parmelee, J.A.; Taylor, R.L., 9.7.1956, (WTU, 197744). British Columbia, Anahim Lake, 1219 m, Calder, J. s.n., Parmelee, J.A.; Taylor, R.L., 9.7.1956, (COLO, 158463). British Columbia, Summit Pass, Raup, H.M. 10788 and Correll, D.S., 24.7.1948, (RM, 272042). Manitoba, Fort Churchill, Ritchie, J. 2104, 5.8.1956, (WIN, 22433). Manitoba, Wapusk National Park, 10 m, Punter, E. 03-509 and Piercey-Normore, M., 19.7.2003, (WIN, 71429). Manitoba, Twin Lakes, Ford, A. 02379, Piercey-Normore, M.; Punter, E.; Punter, D., 25.7.2002, (WIN, 71024). Manitoba, Fort Churchill, Johnson, K. J73-402, 26.8.1973, (WIN, 33557). Manitoba, Fort Churchill, Shay, J. 59-924a, 9.7.1959, (WIN, 64354). Manitoba, Fort Churchill, Shay, J. 83-60, 11.7.1983, (WIN, 40808). Manitoba, Fort Churchill, Zbigniewicz, M. 83-237, 5.8.1983, (WIN, 40839). Manitoba, Wapusk National Park, 15 m, Ford, A. 02-330, Piercey-Normore, M.; Punter, D.; Punter, E., 21.7.2002, (WIN, 70209). Manitoba, Wapusk National Park, 23 m, Ford, A. 02-306, Piercey-Normore, M.; Punter, D.; Punter, E., 20.7.2002, (WIN, 70255). Manitoba, Vicinity of Churchill, Schofield, W. 6862 and Crum, H., 21.7.1956, (CAN, 247332). Manitoba, Fort Churchill, Ritchie, J. 2104, 5.8.1956, (CAN, 248387). Manitoba, Open coastal plain 3 miles East of camp, McFarlane, D.M. 239 and Irvine, B.R., 7.8.1953, (CAN, 322733). Manitoba, Fort Churchill, Brown, D.K. 733, 12.7.1951, (CAN, 263696). Manitoba, Fort Churchill, Argus, G.W. 425-58, 4.8.1958, (CAN, 281144). Manitoba, Fort Churchill, Rossbach, G.B. 7073, 5.8.1965, (CAN, 329753). Manitoba, Fort Churchill, s.n., 30.7.1910, (CAN, 17340). Northwest Territories, Aubry Lake, Riewe, R. 225 and Marsh, J., 17.7.1976, (WIN, 32000). Northwest Territories, Aubry Lake, Riewe, R. 336 and Marsh, J., 4.8.1976, (WIN, 31438). Northwest Territories, Aubry Lake, Riewe, R. 225 and Marsh, G. .M., 17.7.1976, (CAN, 433230). Northwest Territories, Kakisa river, Thieret, J.W. MM3 and Reich, R.J., 18.6.1959, (CAN, 298045). Northwest Territories, Sawmill Bay, Shacklette, H.T. 2970, 13.7.1948, (CAN, 199991). Ontario, Fort Severn, Hustich, I. 1296, 13.7.1956, (CAN, 242845). Ontario, Winisk, Lundsden, H. s.n., (COLO, 448829). Ontario, Kenora District, Riley, J.L. 5848, 23.8.1976, (CAN, 409561). Ontario, Lake River, Dutilly, A. 16550-16807 and Lepage, E., 12.9.1946, (CAN, 17332). Quebec, Fort Chimo, Calder, J. 2316, 2.8.1948, (RM, 216050). Saskatchewan, Hwy #2 Waskesim, Hudson, J. 5063, 31.7.1992, (CAN, 565528). Yukon, Mile 85 on road from Whitehorse to Dawson, 579 m, Calder, J. 25796 and Gillett, J., 22.6.1960, (ALA, 1124987). Yukon, Kluane Lake Quad, 1036 m, Scotter, W. 20992 (Y-18), 2.8.1972, (ALA, 1124986). Yukon, Francis Lake, Duman, G. 70- 805, 28.7.1970, (ALA, 1124985). Yukon, Ogilvie Mountains, Porsild, A.E. 1462, Porsild, R., 28.6.1968, (CAN, 318349). Yukon, Alaska Highway at milepost 1149, Welsh, S.L. 7921, Moore, G., 5.7.1968, (BRY, 71334). Yukon, Rink Rapids, Macoun, 7922, 9.7.1902, (CAN, 17356). Yukon Territory, Dempster Highway, Porsild, R. 1593, 17.7.1968, (CAN, 318505).Finland, Enontekiö Lapland, Lake Raittijärvi, 545 m, Väre, H. 11643, 8.8.2001, (H, 737942). Enontekiön Lappi, Enontekiö, 520 m, Piirainen, M. 2118 and Piirainen, P., 19.7.1991, (H, 668357).

132 ______Chapter 2. Taxonomy of the Carex capitata complex

Enontekiön Lappi, Enontekiö, 600 m, Väre, H. 14955, 1.8.2003, (H, 746021). Enontekiön Lappi, Goaskinjörvi, Kulmala, H. 83/02, 8.8.2001, (H, 744865). Inari Lapland, Kevo Research Station, Sulkinoja, M. s.n., 12.9.1967, (M, 0151936). Inarin Lappi, Kietsimäjoki, Kulmala, H. 8/97, 27.7.1997, (H, 720181). Kainuu, Yli- Näljänkä, 230 m, Ohenoja, M. 11, 8.8.1990, (H, 696101). Karesuando, Karesuando, Honkell, J.s.n., 9.8.1923, (M, 0151934). Kemi Lapland, Vesmajärvi, 210 m, Kurtto, A. 1778, Vuokko, S., 10.8.1978, (O, 660352). Kittilä, Mustavaara, 202 m, Ulvinen, T. s.n., Vilpa, E.; Seitapuro, H., 10.7.1997, (H, 720622). Kuusamo, Liikasenvaara, Ulvinen, T. s.n., 9.8.1962, (O, 539355)., Kuusamo, Liikasenvaara, Ulvinen, T. s.n., 9.8.1962, (M, 0151946). Kuusamo, Lake Paanajärvi, Laurila, M. s.n., 9.7.1938, (H, 272411). Kuusamo, Liikasenvaara, Kukkonen, I. s.n., 30.8.1966, (RMS, 284390). Kuusamo, Liikasenvaara, Ulvinen, T. s.n., 9.8.1962, (CAN, 276804). Kuusamo, NE- section, Paanajärv, Savola, J. s.n., 28.7.1985, (H, 616973). Länsi-Suomen Lääni, Frösön, Mickström s.n., Lagerheim, C.; Sjögren, G., 8.1844, (GH). Lapland, Upper Kemi-river, Ulvinen, T. s.n., 12.8.1961, (C). Lapland, Poroeno, 540 m, Väre, H. 11651, 9.8.2001, (H, 737950). Lapland, Kivijärvi, 460 m, Väre, H. 11515, 29.7.2001, (H, 737814). Lapland, Upper Kemi-river, Ulvinen, T. s.n., 12.8.1961, (H, 328698). Lapland, Tulppio district, Vuokko, S. 8, 29.7.1975, (H, 449415). Lapponia, Muornis, Montell, I. s.n., 17.7.14, (GH). Lapponia, Euvntekiensis, Montell, I. s.n., 9.8.1923, (M, 0151944). Lapponia, Shishe, Montell, I. s.n., 11.7.1909, (M, 0151913). Lapponia, Kouda, Brotherus, V.F. s.n. and Brotherus, A.H., .8.1872, (H, 244602). Lapponia orientalis, Tjavauga, Brenner, M. s.n., 4.7.1863, (H, 1037144). Lapponia Varsugæ, Kihlman, A.O. s.n., 19.8.1889, (H, 328709). Petsamo, Primmanki, Saxén, U. s.n., 13.7.1930, (H, 328729). Pohjanmaa, Ylitornio, Mellakoski, 137 m, Ulvinen, T. s.n., 24.7.1980, (COLO, 394339). Pohjois-Pohjanmaa, Pessalompolo, 140 m, Ulvinen, T. s.n., Karjalahti, T., 30.7.1976, (H, 457472). Sompion Lappi, Petkula, Ohenoja, E. s.n., Melamies, H., 26.7.1996, (H, 722418). Tulijoki, Kainuu, Lehtonen, L. s.n., 18.7.1933, (DAO, 257434). Tulijoki, Kainuu, Lehtonen, L. s.n., 18.7.1933, (DAO, 257433). Tuntsa, Ylitornio, Mellakoski, Kämäräinen, H. 1999- 215, 16.7.1999, (H, 732554). Vaskojoki, Kihlman, A.O. s.n., .8.87, (GH). Germany, Bavaria, Monacho Bavaria, Brügger, C. s.n., 29.6.1873, (GH, 2275). Bavaria, Mikalum, Buccarini s.n., (GH). Bavaria, Oberbayern, Seurs 2053, 27.5.1949, (M, 0151919). Bavaria, Oberbayern, Seurs s.n., 22.5.1851, (M, 0151916). Bavaria, Oberbayern, Leuvs s.n., Seuvnad, 9.6.1851, (M, 0151915). Bavaria, Deining, Brügger, C. 2275, 29.6.1873, (H, 1093339). Oberbayern, Haspelmoor, Holler s.n., 6.1872, (M, 0151918). Oberbayern, Deininger Fliz, Ohmüller s.n., 5.1867, (M, 0151920). Spitzel, V. 379, 1960, (O, 135). Oberschwaben, Schánzle 5.1880, 5.1880, (M, 0151921). Fleischer 18-1900, 1900, (H, 1226126). Greenland, Vestgrønland, Sydostbugten, 80 m, Møller, M. 1156, 15.7.1981, (C).Vestgrønland, Akuliarusikavsak, Jakobsen, K. 12291, 11.8.1956, (C). Iceland, Akureyrense, Skjóldalsárgil, Hg, H. 1529, 20.6.1965, (H, 1226120). Akureyri, Løgumshlid, Grøntved, J. s.n., 24.7.1928, (GH). Árnessýsla, Votamýri, 60 m, Löve, A. A095, Löve, D., 25.9.1949, (GH, 095). Belgsá, Fnjóskadal, Kristinnsson, H. 5143, 27.7.1973, (DAO, 288690). Borgarnes, Borgarnes Fjöfdur, Scamman, E. 1260, 22.8.1938, (GH, 1260). Dalfjall, Mývatnssvei, 460 m, Einarsson, E. E6042, 21.8.1974, (ICEL, 04073). Egilsstaðir, Héraði, 80 m, Meyer, Dr. med. G 7146, 27.8.1932, (ICEL, 04083). Egilsstaðir, Vopnafirði, Stefánsson I, S. 256, 4.8.1895, (ICEL, 04088). Finnsstaðir, Eiðaþinghá, Lagarfljótsrannsóknir 7145, 24.7.1975, (ICEL, 04082). Hallormsstadur, Egilsstadir, Gøtzsche, H.F. 81.37, 22.7.1981, (C, 8). Hrísey, Eyjafirði, Garðarsson, A. s.n., 12.8.1967, (ICEL, 04078). Hrísey, Eyjafirði, Garðarsson, A. s.n., 8.8.1967, (ICEL, 04077). Hvalfjörður, Ingimarsson, Ó. s.n., 11.8.1951, (DAO, 257458). Lagarfoss, Hróarstungu, Lagarfljótsrannsóknir s.n., 26.6.1976, (ICEL, 04080). Lagarfoss, Fljótsdals, Magnússon, S.H. s.n., 26.6.1976, (ICEL, 47380). Lagarfoss, Hróarstungu, Lagarfljótsrannsóknir s.n., 26.6.1976, (ICEL, 04081). Moldhaugar, Kræklingahlíð, Óskarsson, I. 935, 22.8.1926, (ICEL, 04052). Nes, Höfðahverfi, Óskarsson, I. 681, 30.6.1926, (ICEL, 46472). Öræfi, Bæjarstaðarskógur Öræfum, Björnsson, H. 9633, 16.10.1947, (ICEL, 04074). Öræfi, Fagurhólsmýri Öræfum, Björnsson, H. 9638, 7.1947, (ICEL, 04075). Öræfi, Skaftafell Öræfum, Björnsson, H. 9624, 15.6.1946, (ICEL, 04076). Reykjahlið, Lake Mývatn, 280 m, Seberg, O. 427, 14.8.1976, (C, 7). Sellátur, Reyðarfirði, Óskarsson, I. s.n., 14.7.1927, (ICEL, 04051). Skagafjord, Valnsfjall, Sørensen, T.H. 31/7, 31.7.1930, (O, 539367). Vaglaskógur, Fnjóskadal, Óskarsson, I. 1295, 7.8.1927, (ICEL, 04055). Vesturdalur, Bachufer, Lang, W. s.n., 29.7.1987, (M, 0151937). s.n., 9.7.87, (GH, ). 285 m, Lid, J. s.n., 14.7.1937, (O, 539360). Italy, South Tirol, Seiser Alm, 1860 m, Bestand, G. s.n., 17.7.1958, (M, 0151928). South

133 ______Chapter 2. Taxonomy of the Carex capitata complex

Tirol, 1100 m, Hoock, G. s.n., 3.8.1908, (M, 0151922). South Tirol, Seisseralpe, , Koch, J. s.n., 7.7.1955, (M, 0151914). South Tirol, Seiser Alm, 1900 m, Hertel, H. 4324, 27.6.1964, (M, 0151927). South Tirol, Dolomiten, 2370 m, s.n., 15.7.1958, (M, 0151924). South Tirol, Seisseralpe, 1980 m, Roessler, H. 2519, 25.7.1959, (M, 0151925). South Tirol, Seiser Alm, 2000 m, Dietrich, W. 3283, 28.6.1964, (M, 0151930). South Tirol, Bozen, 1950 m, Dietrich, W. 1963-66, 28.6.1964, (M, 0151929). Südtirol, Feuchstelle, 2200 m, Angerer,O. s.n., 23.7.1976, (M, 0151926). Norway, Finnmark, Veinesbukt, Skifte, O. s.n., Stellander, O., 6.8.1967, (C). Finnmark, Kautokeino, 340 m, Kautokeino, N. s.n., Mieron, N.; Moor, 23.8.1967, (M, 0151938). Finnmark, Bugöynes, 20 m, Toivonen, H. s.n., 3.8.1977, (H, 1471327). Finnmark, Bugöynes, 20 m, Toivonen, H. s.n., 3.7.1977, (H, 1471326). Finnmark, Bugöynes, 25 m, Toivonen, H. s.n., 3.8.1977, (H, 1468929). Finnmark, Billefjord, 5 m, Toivonen, H. s.n., 1.8.1972, (H, 1470511). Hamar, Jerkim, Conradi, F.E. s.n., 15.7.1887, (GH). Hedmark, Gammelsetran, 860 m, Vileid, M. s.n., 18.8.1998, (O, 235091). Hedmark, Jogåsmyra, 630 m, Kielland-Lund, J. s.n., 9.7.1967, (O, 176158). Hedmark, Os, 780 m, Elven, R. s.n., (O, 4689). Hedmark, Folldal, 840 m, Buttle 8066, Gauhl, 19.8.1965, (M, 0151912). Hjerkinn, Stanley Pease, A. s.n., 21.7.1930, (GH, 20740). Hordaland, Eidfjord, 100 m, Lid, J. s.n., 26.7.1936, (O, 414980). Kongsvold, Dovrefjeld, Nilsson, S.J. s.n., .8.1898, (GH). Nordland county, Sørfold, Apold, W. s.n., Brodal, G.; Skifte, O., 8.8.1954, (H, 1013890). Norland, Nordland fylke, , Notø, A. s.n., 6.7.1932, (M, 0151945). Oppland, Espedal, Berg, R.Y. s.n., 11.8.1973, (O, 260563). Oppland, Lom, 940 m, Berg, R.Y. s.n., 10.8.1994, (O, 174746). Oppland, Grimsdalen, 900 m, Bratli, H. s.n., 28.7.1994, (O, 114994). Sör-Tröndelag, Opdal herred, Kongsvoll, Nilsson, S.J. s.n., .7.1883, (DAO, 257470). Sör-Tröndelag, Oppdal, Near Kongsvoll, Wendelbo, P. s.n., 17.7.1948, (COLO, 100223). Troms, Stordalen, 250 m, Engelskjøn, T. s.n., 24.7.1962, (C). Troms, Lulleborg, 360 m, Lye, K.A. 18728, Berg, T., 1.9.1992, (O, 75397). Troms, Fossbakken, Svendsen, S. s.n., 31.7.1967, (O, 92610). Tromsö, Ringvatso Island, 30 m, Notø, A. s.n., 10.7.1896, (GH). Russia, Chita region, Between the rivers Nerchei and Kuengoi, Sukatschew, W. s.n., 10.7.1911, (DAO, 142005). Chukotka national district, Anui upland region, Zimarskaja, E.V. s.n., Korobkov, A.A.; Yurtsev, B.A., 12.7.1967, (DAO, 139880). Chukotka national district, Rauchua river, Yurtsev, B.P. s.n., 12.7.1967, (BRY, 122530). Chukotski peninsula, river Utaveem, , Kozhevnikov, U.P. s.n., Nechaev, A.A.; Yurtsev, B.A., 27.7.1970, (COLO, 323093). Irkutsk, Balagansk region, Maltsev, I. s.n., 19.6.1905, (GH). Kamchatka region, Olyutorsky area, Harkevich, S. s.n., 9.8.1975, (GH). Komi Republic, Syktyvkar, Andreev, V.D. s.n., 21.6.1909, (H, 1037137). Magadan region, North Even, Hohrjakov, A.P. s.n., 2.8.1976, (CAN, 455497). Republic of Karelia, Karelia onegensis (Kon), Ruuhijärvi, R. 40/02, 9.7.2002, (H, 744530). Republic of Karelia, Belomorskiy District, 10 m, Kravchenko, A. s.n., 21.8.2002, (H, 742280). Republic of Karelia, Karelia pomorica orientalis, Piirainen, M. 5376, 19.8.2004, (H, 807345). Republic of Karelia, Karelia pomorica orientalis, 20 m, Piirainen, M. 5027, 22.8.2002, (H, 741569). Sakha Republic, Bulunsk region, Yurtsev, B.A. s.n., 25.6.1960, (DAO, 257437). Taymyr, River Pyasina, Kozhevnikov, U.P. s.n., 21.8.1982, (CAN, 490439). Between the rivers Nerchei and Kuengoi, Sukachev, V. s.n., 27.7.1970, (DAO, 139887). Kihlman, A.O. s.n., 18.8.1891, (H, 1226124). Chersky, Kozhevnikov, U.P. 714, 24.7.1977, (CAN, 455526). Sweden, Dalecarlia, Morängen, Källström, S. s.n., 7.1887, (GH). Dalecarlia, Fries s.n., (GH). Härjedalen, Valmåsen, Dusén, K. s.n., 11.8.1879, (DAO, 363985). Jämtland, Paroecia Frösö, Asplund, E. s.n., 2.6.1925, (GH). Jämtland, Paroecia Frösö, Asplund, E. s.n., (C). Jämtland, Nyhem, 280 m, s.n., 4.7.1977, (M, 0151939). Jämtland, Häggenås, 400 m, s.n., 3.7.1977, (M, 0151940). Jämtland, Mosjön, 305 m, s.n., 5.7.1977, (M, 0151941). Jämtland Ås, Ahlqvist, A. s.n., 28.6.1902, (GH). Kilpisjärvi, Saana, 50 m, Roivainen, L. s.n., 14.7.1958, (DAO, 257436). Lule Lappmark, Avvakko- tunturi, 500 m, Hertel, H. 7248b, 21.7.1967, (M, 0151935). Scandinavia, s.n., 1887, (M, 0151938). Sverige, Torne Lappmark, Pederson, T.M. 5615, 15.7.1960, (O, 314293). Torne Lappmark, Torne Träsk, Torlöf, A. s.n., 12.8.1958, (GH). Torne Lappmark, Låktatjakko, 700 m, Alm, G. 449, 11.8.1935, (GH, 449). Torne Lappmark, Jukkasjärvi, 333 m, Alm, G. s.n., 8.8.1935, (GH, 442). Torne Lappmark, Abisko, Selander, S. s.n., 9.7.1905, (GH). Torne Lappmark, Lake Torneträsk District, 450 m, Alm, G. s.n., 9.8.1958, (O, 539346). Torne Lappmark, Abisko, Hertel, H. 22918, 8.8.1980, (M, 0151931). Torne Lappmark, Abisko, s.n., 13..8, (M, 0151947). Torne Lappmark, Abisko, Hiitonen, I. s.n., 22.7.1950, (H, 1693670). Torne Lappmark, Abisko, 400 m, Alm, G. s.n., 6.8.1958, (H, 1226056). USA, Alaska, Old John Lake Area, Holmen, K. 61-1227, 13.7.1961, (C, 61-1227).

134 ______Chapter 2. Taxonomy of the Carex capitata complex

Alaska, Wiseman, Anderson, J.F. 5970, Gasser, G.W., 3.8.1939, (ALA, 1125027). Alaska, Shaw Creek Flats, Elven, R. s.n., Solstad, H., 28.7.2001, (ALA, 1125006). Alaska, Euchre Moutain, 3868 ft, Bennett, B. 194/13273, Loomis, P., 20.6.2003, (ALA, 1125007). Alaska, Smith Lake, Parker, C.L. 15339, 7.8.2003, (ALA, 1125008). Alaska, Central Noatak R. Valley, 100 m, Parker, C.L. 15128, Elven, R.; Solstad, H., 23.7.2003, (ALA, 1124990). Alaska, Kilikmak Cr., 8 m, Parker, C.L. 14722, Elven, R.; Solstad, H., 13.7.2003, (ALA, 1124991). Alaska, Mt. Hayes, 419 m, Duffy, M. 98-201, 15.7.1998, (ALA, 1124993). Alaska, Endicott Mountains, 900 m, Parker, C.L. 12108, Elven, R.; Solstad, H.; Bennett, B.A., 19.7.2002, (ALA, 1124994). Alaska, Neacola Moutains, Caswell, P. 96-205, 19.6.1996, (ALA, 1124995). Alaska, Mt. Michelson, 861 m, Batten, A. 686, 26.7.1973, (ALA, 1124996). Alaska, Howard Pass, 700 m, Parker, C.L. 7648, , 27.7.1997, (ALA, 1124998). Alaska, Table Mountain, 622 m, Mouton, M.A. MM79279, 30.6.1979, (ALA, 1125000). Alaska, Imiaknikpak Lake, 581 m, Murray, D.F. 4314, 27.7.1973, (ALA, 1124970). Alaska, Baird Mountains, 85 m, Parker, C.L. 15299, Elven, R.; Solstad, H., 29.7.2003, (ALA, 1124972). Alaska, Charley River, 850 ft, Larsen, A. 02-2430, Batten, A., 25.7.2002, (ALA, 1124973). Alaska, Bering Land Bridge NPreserve, 250 m, Kelso, T. 87-319, 7.7.1987, (ALA, 1124975). Alaska, McKinley River, 1900 ft, Viereck, L.A. 1613, 30.7.1956, (ALA, 1124982). Alaska, Arctic National Wildlife Range, 430 m, Murray, D.F. 3350, 26.7.1970, (ALA, 1124984). Alaska, Alaska Range, 750 m, Duffy, M. MD02-240, 16.8.2002, (ALA, 1125011). Alaska, Alaska Range, 725 m, Roland, C. 4519, Batten, A.; Goeking, S., 7.1.2000, (ALA, 1125012). Alaska, Solomon, 85 m, s.n., 14.7.2000, (ALA, 1125013). Alaska, Seward Peninsula, 37 m, Murray, D.F. 11077, Yurtsev, B.A.; Kelso, T., 26.7.1992, (ALA, 1125015). Alaska, Kokrine Hills, 275 m, Foote, J. JF4208, 24.6.1980, (ALA, 1125016). Alaska, Fort Wainwright Military Reservation, 115 m, Duffy, M. 95-624, Lipkin, R., 10.7.1995, (ALA, 1125017). Alaska, Jago Lake, Cantlon, J.E. 57- 1613, Gillis, W.T., 28.7.1957, (ALA, 1125021). Alaska, Tanana River, Spetzman, L. 11868, 7.8.1957, (ALA, 1125022). Alaska, Bendeleben Quad, 100 m, Kelso, T. 82-190, 10.8.1982, (COLO, 387320). Alaska, Mt. Mckinley Natl. Park Teklanika River, 792 m, Viereck, L.A. 7427, 3.8.1964, (ALA, 1125025). Alaska, Mt. Mckinley Natl. Park Teklanika River, 792 m, Viereck, L.A. 7427, 3.8.1964, (RMS, 430206). Alaska, Mt. Mckinley Natl. Park Teklanika River, 792 m, Viereck, L.A. 7427, 3.8.1964, (CAN, 362141).

Studied specimens of C. cayoutteana subsp. cayouetteana Snow Creek Pass, 7400 ft, Calder, J. 23957, 24.7.1959, (COLO, 148926). Alberta, Snow Creek Pass, 7000 ft, Porsild, A.E. 22673, 29.7.1960, (RM, 529780). British Columbia, Bluster Mt., 2133 m, Thompson, J. s.n., Thompson, M., 14.7.1938, (WTU, 48964). British Columbia, Chipuin Mt., 1828 m, Thompson, J. s.n., Thompson, M., 21.7.1938, (WTU, 17893). British Columbia, Quiniscoe Lake, 2316 m, Calder, J. 19594, Parmelee, J.A.; Taylor, D., 2.8.1956, (WTU, 199618). British Columbia, 7100 ft, Calder, J. 11795, Savile, O., 11.8.1953, (COLO, 118024). British Columbia, Ashnola Range, 7600 ft, Calder, J. 19594, Parmelee, J.A.; Taylor, R.L., 2.8.1958, (RM, 260491). Mexico, Pacheco, Chihuahua, Hartman, C.V. s.n., 10.6.1891, (MICH, 1132452). USA, California, Anderson Mdw., 1950 m, Gierisch, R. 3493, Esplin, D., 25.6.1969, (RMS, 430207). California, Anderson Mdw., 6400 ft, Gierisch, R. 3493, Esplin, D., 25.6.1969, (COLO, 246761). California, Anderson Mdw., Gierisch, R. 3493, Esplin, D., 25.6.1969, (CAS, 690732). California, South Warner Mountains, 9000 ft, Otting, N. NAD27, Lytjen, D., 2.9.2004, (OSC, 219450). Colorado, Bill Moore Lake, 3627 m, Lederer, N. 4257, 31.8.1993, (COLO, 00263731). Colorado, Loch Lomond, 3395 m, Weber, W.A. s.n., Koponen, T.; Nelson, P., 8.8.1972, (CAN, 374041). Colorado, San Juan National Forest, 11900 ft, Rink, G. 3668, 25.7.4, (BRY, 467234). Colorado, Loch Lomond, 11140 ft, Weber, W.A. s.n., Koponen, T.; Nelson, P., 8.8.1972, (COLO, 259883). Colorado, Hagerman Pass, 11980 ft, Hartman, E.L. 6718, Rottman, M.L., 29.8.1986, (COLO, 428741). Colorado, Fraser Exp. Forest, 12000 ft, Weber, W. 8621, Dahl, E., 31.7.1953, (COLO, 76204). Colorado, Mesa Seco, 12300 ft, Johnson, K. J64-117, (COLO, 232659). Montana, Sweet Grass County, 2956 m, Lesica, P. 7663, 27.7.1998, (MONTU, 122991). Montana, Sweet Grass County, 2743 m, Lesica, P. 7362, 10.8.1996, (MONTU, 122399). Montana, Sweet Grass County, 2743 m, Lackshewitz, H. 9909, 15.8.1981, (MONTU, 86558). Montana, Carbon County,

135 ______Chapter 2. Taxonomy of the Carex capitata complex

2999 m, Ramsden, J. 1625, 10.7.1987, (MONTU, 118978). Montana, Carbon County, 2987 m, Lesica, P. 5583, 15.8.1991, (MONTU, 115081). Montana, Carbon County, 3048 m, Lesica, P. 4483, 11.8.1987, (MONTU, 108435). Montana, Park County, Ramsden, J. 85542, 9.7.1980, (MONTU, 85542). Montana, Carbon County, 3017 m, Lackshewitz, H. 7790, 11.8.1977, (MONTU, 78793). Montana, Sweet Grass County, 2743 m, Lackshewitz, H. s.n., 15.8.1981, (COLO, 355226). Montana, Stillwater County, 2767 m, Evert, E. 24076, 27.7.1992, (RMS, 780026). Montana, Carbon County, 3048 m, Evert, E. 19835, 23.7.1990, (RMS, 619855). Montana, Carbon County, 2987 m, Lackshewitz, H. 7000, 14.9.1976, (WTU, 272540). Montana, Carbon County, 3017 m, Lackshewitz, H. 7790, 11.8.1977, (WTU, 288770). Montana, 9800 ft, Lackschewitz, K.H. 7035, 15.9.1976, (RM, 367206). Montana, Beartooth Pass, 11000 ft, Hermann, F.J. 20079, 20.7.1965, (RMS, 430211). Montana, 9000 ft, Evert, E. 18434, 9.8.1989, (RM, 579301). Montana, Lackschewitz, K.H. 9909, 15.8.1981, (RM, 521779). Montana, 9900 ft, Lackschewitz, K.H. 7790, 11.8.1977, (RM, 367094). Montana, Sweet Grass County, 9000 ft, Lackschewitz, K.H. s.n., 15.8.1981, (GH). Nevada, Browns Cr., 2590 m, Lewis, E. 448, 17.7.1955, (RMS, 390545). Nevada, Browns Cr., 2590 m, Lewis, E. 17.7.1955, (CAN, 550536). Utah, Uinta Mountains, Lewis, E. 512, 15.8.1955, (RMS, 368032). Utah, Gilbert Bench, 3505 m, Goodrich, S. 25583, Huber, A.; Prescott, D., 20.8.1996, (BRY, 392186). Utah, Gilbert Creek, 3493 m, Huber, A. 440, Goodrich, S., 25.8.1993, (BRY, 368578). Utah, Uinta Mountains, Lewis, E. 512, 15.8.1955, (CAN, 515168). Utah, Gilbert Bench, 12100 ft, Goodrich, S. 26303, Huber, A.; Frandsen, J.; Bartlett, F., 9.8.2000, (BRY, 437123). Utah, Ridge saddle, 12600 ft, Huber, A. 4134, 3.8.1999, (BRY, 426752). Utah, Ashley Forest, 11850 ft, Goodrich, S. 23530, Bartlett, F.; Atwood, D.; Nelson, D., 19.8.1991, (BRY, 350794). Washington, Chowder Ridge, 6800 ft, Douglas, G. 4345, Douglas, G., 3.8.1972, (DAO, 621358). Washington, Rocky Mt., 2365 m, Douglas, G. 2887, 19.7.1971, (RMS, 430209). Wyoming, 10700 ft, Mosquin, T. 4817, 2.8.1962, (DAO, 257425). Wyoming, 3279 m, Mellmann-Brown, S. 2575, 7.8.1996, (RMS, 644114). Wyoming, Elk Peak, 3566 m, Hartman, L. 24223, Poll, T., 9.8.1988, (RMS, 533361). Wyoming, 3474 m, Hartman, L. 31265, 19.8.1991, (RMS, 589096). Wyoming, Neely, B. 2435, 18.8.1984, (COLO, 399492). Wyoming, Beartooth Plateau, 3300 m, Weber, W. s.n., 18.8.1973, (COLO, 270915). Wyoming, Beartooth Plateau, 9800 ft, Lackshewitz, H. s.n., 14.9.1976, (COLO, 306544). Wyoming, Cascade Creek, 10300 ft, Evert, E. 18305, 3.8.1989, (COLO, 449077). Wyoming, Lamar River, 10300 ft, Nelson, B.E. 12725, Hartman, R.L., 22.7.1985, (RM, 482304). Wyoming, Beartooth Plateau, 9800 ft, Lackschewitz, K.H. 7000, 14.9.1976, (RM, 367209). Wyoming, Beartooth Plateau, 9800 ft, Dorn, R.D. 3590, 12.8.1980, (RM, 330260). Wyoming, Francs Fork, 11150 ft, Hartman, L. 16805, 14.8.1983, (RM, 558454). Wyoming, Beartooth Plateau, 10800 ft, Mellmann-Brown, S. 2470, 22.7.1996, (RM, 612812). Wyoming, Eastern Wind River Range, 10240 ft, Mills, S. 232a, 18.8.1995, (RM, 603492). Wyoming, Head Elk Creek, 11500 ft, Johnson, W.M. 140, 29.8.1961, (RMS, 401425). Wyoming, Northern Wind River Range, 10240 ft, Mills, S. 230b, 18.8.1995, (RM, 603491). Wyoming, Bug Creek Pass, Absarokas, 11000 ft, Johnson, W.M. 270, 8.8.1962, (RM, 189438-s). Wyoming, Bug Creek Pass, Absarokas, 11000 ft, Johnson, W.M. 270, 8.8.1962, (RMS, 401298). Wyoming, Absaroka Mountains, 10000 ft, Kirkpatrick, R.S. 5901, Kirkpatrick, R.E.B., 14.8.1984, (RM, 558456). Wyoming, Cascade Creek, 10300 ft, Evert, E. 18305, 3.8.1989, (RM, 579204). Wyoming, Absaroka Mountains, 11150 ft, Kirkpatrick, R.S. 5910, Kirkpatrick, R.E.B., 21.8.1984, (RM, 558455). Wyoming, Absaroka Mountains, 10200 ft, Evert, E. 18249, 3.8.1989, (RM, 579080). Wyoming, Absaroka Mountains, 9800 ft, Evert, E. 9608, 20.8.1985, (RM, 623052). Wyoming, Absaroka Mountains, 10000 ft, Hartman, R.L. 19105, 21.8.1984, (RM, 558453). Wyoming, Absaroka Mountains, 11750 ft, Hartman, R.L. 19289, 22.8.1984, (RM, 558452). Wyoming, Absaroka Mountains, 10700 ft, Hartman, R.L. 23927, Poll, T., 5.7.1988, (RM, 536641). Wyoming, West Slope Wind River Range, 10400 ft, Hartman, R.L. 31278, 19.8.1991, (RM, 589095). Wyoming, Absaroka Mountains, 10500 ft, 4416, 20.7.1984, (RM, 558457). Wyoming, Beartooth Plateau, 9570 ft, Fertig, W. 15202, 23.7.1994, (RM, 602345). Wyoming, Mellmann-Brown, S., 24.8.1996, (RM, 615036).

Studied specimens of C. cayouetteana subsp. bajasierra

136 ______Chapter 2. Taxonomy of the Carex capitata complex

USA, California, El Dorado Co., Echo Summit, Howell, J.T. 257424, , 1.9.1946, (DAO, 257424). California, El Dorado Co., El Dorado National Forest, 2350 m, Toivonen, H. 661914, Norris, D.H.; Pykälä, J., 23.7.1987, (DAO, 661914). California, El Dorado Co., El Dorado National Forest, 2350 m, Pykälä, J. 6, Norris, D.H.; Toivonen, H., 23.7.1987, (C, 6). California, El Dorado Co., Freel Peak quad, 2292m, Janeway, L. 73322, Schroder, E., 2.9.1998, (CHSC, 73322). California, Plumas County, Blucks Lake quad, 481 m, Janeway, L. 78722, 7.7.2000, (CHSC, 78722). California, Tehama County, Yellow Pine Forest, 1540 m, Ahart, L. 94326, 19.7.2006, (CHSC, 94326). California, Sierra County, Yuba Pass- Weber Lake Rd., 2194 m, Oswald, H. 66824, Ahart, L., 19.8.1996, (CHSC, 66824). California, Nevada County, University of California Trout Lab, 6500 ft, Langenheim, J. 272099, 19.7.1957, (CAN, 272099). California, Nevada County, University of California Trout Lab, 6500 ft, Nisbet, W.A. 272091, 20.7.1957, (CAN, 272091). California, Nevada County, Sagehen Creek, 6300 ft, True, G.H. 845706, Howell, J.T., 29.8.1966, (CAS, 845706). California, Nevada County, University of California Trout Lab, 6500 ft, Langenheim, J. 845707, 19.7.1957, (CAS, 845707). California, Lassen Volcanic National Park, Badger Flat, 6275 ft, Leschke, H. 136120, 10.8.1960, (OSC, 136120). California, Nevada County, Truckee, 2035 m, Naczi, R.F.C., 3.8.2006, (NYBG). California, Nevada County, Truckee, 1980 m, Naczi, R.F.C., 4.8.2006, (NYBG). California, Tulare County, Kaweah Meadows, Howell, J.T. 17724, 5.8.1942, (GH, 17724). Oregon, Lake County, Sycan Marsh, 1524 m, Christy, A. 188302, 23.8.1980, (OSC, 188302). Oregon, Deschutes County, 1981 m, Wilson, B. 178855, 9.8.1990, (OSC, 178855). Oregon, Jackson County, Cascade Mountains, 1636 m, Otting, N. 210656, 28.6.2001, (OSC, 210656). Oregon, Deschutes County, 1926 m, Halpern, C. 159046, Magee, T., 30.8.1982, (OSC, 159046).

Studied specimens of C. cayouetteana subsp. altasierra

USA, California, Tulare Co., Sierra Nevada, 12000 ft, Howell, J.T. s.n., 5.8.1949, (DAO, 257423). California, Inyo County, Mount Humphreys, 12880 ft, Sharsmith, C.W. 3116, 11.8.1937, (DAO, 257428). California, Inyo County, Mono Mesa, 3657 m, Howell, J.T. s.n., 26.7.1946, (WTU, 137524). California, Mono County, Mt. Dana Plateau, 3505 m, Taylor, D. 7550, 25.7.1979, (COLO, 330874). California, Sierra Nevada, Central Basin, 3444 m, Munz, A. 12669, 26.7.1948, (WTU, 133536). California, Tuolumne County, Kuna Peak, 12500 ft, Sharsmith, C.W. 2681, 21.7.1937, (CAN, 162869). California, Mono County, White Mountains, 11800 ft, Morefield, J.D. 4829, Perala, C., 27.7.1988, (MICH). California, Mono County, Dunderberg Peak, 11800 ft, Taylor, D. 5291, 27.7.1975, (CAS, 856994). California, Fresno County, 11192 ft, Quibell, C.H. 4162, 7.8.1954, (OSC, 96143). California, Inyo County, Mono Mesa, 12000 ft, Howell, J.T. s.n., 26.7.1946, (GH, 12750).

137 ______Chapter 2. Taxonomy of the Carex capitata complex

APPENDIX S2

Figure A.1: Histograms of the six discrete variables scored for the morphometric study. C. capitata (C), C. arctogena (A), C. cayouetteana subsp. cayouetteana (Y), C. cayouetteana subsp. bajasierra (Y2) and C. cayouetteana subsp. altasierra (Y3). X axis represents the measurements and Y axis the number of specimens.

138 ______Chapter 2. Taxonomy of the Carex capitata complex

139 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure A.2: Boxplots showing mean interspecific differences between C. capitata (C), C. arctogena (A), C. cayouetteana subsp. cayouetteana (Y), C. cayouetteana subsp. bajasierra (Y2) and C. cayouetteana subsp. altasierra (Y3) for twenty two quantitative and continuous variables. Asterisks to the left of each box denote level of statistical significance based on a Kruskal-Wallis ANOVA. Blue denotes P<0.01 and red P<0.05. Characters are alphabetically arranged. A full description of the characters is given in Table 2.5.

140 ______Chapter 2. Taxonomy of the Carex capitata complex

141 ______Chapter 2. Taxonomy of the Carex capitata complex

142 ______Chapter 2. Taxonomy of the Carex capitata complex

143 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure A.3: Pistillate scales (above) and periginia (below) of C. capitata (C), C.arctogena (A), C. cayouetteana subsp. cayouetteana (Y), C. cayouetteana subsp. bajasierra (Y2) and C. cayouetteana subsp. altasierra (Y3).

144 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure A.4: Holotype of C. capitata L. at LINN.

145 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure A.5: Holotype of C. arctogena Harry Sm. at DAO

146 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure A.6: Holotype of C. antarctogena Roivanen at H.

147 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure A.7: The distribution of C. capitata (circles), C. arctogena (squares), C. cayouetteana subsp. cayouetteana (triangles), C. cayouetteana subsp. bajasierra (crosses) and C. cayouetteana subsp. altasierra (stars) based on all the herbarium specimens examined in this study.

148 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure A.8: The distribution of C. capitata (circles), C. arctogena (squares), C. cayouetteana subsp. cayouetteana (triangles), C. cayouetteana subsp. bajasierra (crosses) and C. cayouetteana subsp. altasierra (stars) in North America based on all the herbarium specimens examined in this study.

149 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure A.9: The distribution of Carex capitata and C. arctogena in Europe based on all specimens examined in this study.

150 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure A.10: The distribution of C. cayouetteana subsp. cayouetteana based on all the herbarium specimens examined in this study.

151 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure A.11: The distribution of C. cayouetteana subsp. bajasierra based on all the herbarium specimens examined in this study. .

152 ______Chapter 2. Taxonomy of the Carex capitata complex

Figure A.12: The distribution of C. cayouetteana subsp. altasierra based on all the herbarium specimens examined in this study.

153 ______Chapter 2. Taxonomy of the Carex capitata complex

154 Chapter 3

Direct long-distance dispersal best explains the bipolar distribution of Carex arctogena (Carex sect. Capituligerae, Cyperaceae)

155 156 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena Journal of Biogeography (J. Biogeogr.) (2015)

ORIGINAL Direct long-distance dispersal best ARTICLE explains the bipolar distribution of Carex arctogena (Carex sect. Capituligerae, Cyperaceae) Tamara Villaverde1*, Marcial Escudero2, Santiago Martın-Bravo1, Leo P. Bruederle3, Modesto Luceno~ 1 and Julian R. Starr4,5

1Botany area, Department of Molecular ABSTRACT Biology and Biochemical Engineering, Pablo Aim The bipolar disjunction, a biogeographical pattern defined by taxa with a de Olavide University, 41013 Seville, Spain, 2 distribution at very high latitudes in both hemispheres (> 55° N; > 52° S), is Department of Integrative Ecology, Estacion Biologica de Donana~ (EBD – CSIC), 41092 only known to occur in about 30 vascular plant species. Our aim was to use Seville, Spain, 3Department of Integrative the bipolar species Carex arctogena to test the four classic hypotheses proposed Biology, University of Colorado Denver, to explain this exceptional disjunction: convergent evolution, vicariance, moun- Denver 80217–3364, USA, 4Canadian tain-hopping and direct long-distance dispersal. Museum of Nature, Ottawa K1P 6P4, Location Arctic/boreal and temperate latitudes of both hemispheres. Canada, 5Department of Biology, Gendron Hall, University of Ottawa, Ottawa K1N 6N5, Methods A combination of molecular and bioclimatic data was used to test Canada phylogeographical hypotheses in C. arctogena. Three chloroplast markers (atpF–atpH, matK and rps16) and the nuclear ITS region were sequenced for all species in Carex sections Capituligerae and Longespicatae; Carex rupestris, C. obtusata and Uncinia triquetra were used as outrgroups. Phylogenetic rela- tionships, divergence-time estimates and biogeographical patterns were inferred using maximum likelihood, statistical parsimony and Bayesian inference.

Results Carex sections Capituligerae and Longespicatae formed a monophyletic group that diverged during the late Miocene. Two main lineages of C. arctoge- na were inferred. Southern Hemisphere populations of C. arctogena shared the same haplotype as a widespread circumboreal lineage. Bioclimatic data show that Southern and Northern Hemisphere populations currently differ in their ecological regimes. Main conclusions Two of the four hypotheses accounting for bipolar dis- junctions may be rejected. Our results suggest that direct long-distance dis- persal, probably southwards and mediated by birds, best explains the bipolar *Correspondence: Tamara Villaverde, Botany area, Department of Molecular Biology and distribution of C. arctogena. Biochemical Engineering, Pablo de Olavide Keywords University, Ctra. de Utrera km 1 s/n, 41013 Seville, Spain. Biogeography, bipolar distribution, Capituligerae, Carex, climatic niche, E-mail: [email protected] Cyperaceae, divergence-time estimation, long-distance dispersal.

century (e.g. Darwin, 1859). However, resolving the biogeo- INTRODUCTION graphical and evolutionary origins of bipolar taxa has been Arctic taxa are often widely distributed, their distributions challenging due to the scale of their distributions. Four main usually fitting into one of three patterns: circumpolar, amphi- mechanisms have been proposed to account for bipolar taxa: Atlantic or amphi-Beringian. When Arctic taxa also occur at (1) vicariance (Du Rietz, 1940), implying fragmentation of a very high latitudes in the Southern Hemisphere (> 52° S), continuous distribution that would date back to the trans- they achieve what is known as a bipolar distribution (Moore tropical highland bridges of the Mesozoic (c. 195 million years & Chater, 1971). This remarkable biogeographical pattern ago, Ma; Scotese et al., 1988); (2) convergent or parallel evo- provides some of the greatest biological disjunctions known lution of disjunct populations that have independent origins and it has inspired authors in biogeography since the 19th but similar phenotypes through adaptation to comparable

ª 2015 John Wiley & Sons Ltd http://wileyonlinelibrary.com/journal/jbi 1 doi:10.1111/jbi.12521 157 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena T. Villaverde et al. environmental pressures (Scotland, 2011); (3) stepwise long- rex magellanica Lam., cool, short-day conditions are suffi- distance dispersal across the equator via mountain ranges cient to induce flowering. The few molecular studies that (‘mountain-hopping’, Moore & Chater, 1971; Heide, 2002; have focused on bipolar Carex are consistent with Heide’s Vollan et al., 2006) during the last cold periods of the Pliocene (2002) results. Both Vollan et al. (2006) and Escudero et al. and Pleistocene that extended the polar regions of both hemi- (2010) found low levels of genetic differentiation in five of spheres (Raven, 1963); and (4) direct long-distance seed dis- the six known bipolar species of Carex, suggesting that either persal by birds, wind or ocean currents (Nathan et al., 2008; mountain-hopping or direct long-distance dispersal was the and references therein). These hypotheses can now be tested best explanation for the species’ current distributions. How- objectively by examining the distribution of haplotypes and by ever, neither Vollan et al. (2006) nor Escudero et al. (2010) dating molecular phylogenies to better assess the possible could determine definitively which hypothesis best explained evolutionary, climatic and geological changes at the origin of the distributions of bipolar species. The only remaining biogeographical patterns (Crisp et al., 2011). bipolar Carex not to have been studied using molecular Most recent studies addressing the origin of bipolar plants markers is Carex arctogena Harry Sm. (in Carex sect. Capit- have focused on supraspecific groups (e.g. Euphrasia, Gussar- uligerae Kuk.),€ a species that reaches both the Canadian ova et al., 2008; Empetrum, Popp et al., 2011) and used Arctic Archipelago in the Northern Hemisphere and the molecular data only. Nonetheless, these studies estimated southernmost region of South America, Tierra del Fuego that the divergence of bipolar lineages occurred a maximum (Fig. 1). Carex sect. Capituligerae includes two other species: of 10 million years ago, and concluded that the best explana- the alpine Carex oreophila C. A. Mey, a species confined to tion for bipolar distributions was long-distance dispersal. Of the mountains of south-western Asia, and the circumboreal the approximately 30 bipolar vascular species that are known Carex capitata L. (Egorova, 1999). (Moore & Chater, 1971), six are found in Carex L., a diverse Although morphological, ecological and molecular data genus (> 2000 species) that is most common in the cold and clearly separate C. arctogena from its sister species, C. capitata, temperate regions of the Northern Hemisphere (Reznicek, in northern Europe (Reinhammar, 1999; Reinhammar & Bele, 1990). Because most Carex species, and especially the bipolar 2001), these differences are less clear in North America, where species, live under long-day conditions, Heide (2002) tested these species are considered to form a complex (Murray, whether the plants could reproduce under the short-day con- 2002). Ecological factors could be influencing the geographical ditions seen in the tropics, in an attempt to refute the distribution of C. arctogena and C. capitata and may therefore hypothesis of trans-equatorial mountain-hopping. Heide’s constitute a key element in determining their distributional results showed that, at least for Carex canescens L. and Ca- patterns. The integration of phylogeographical inferences from

Figure 1 Distribution map of sampled populations of species in Carex sections Capituligerae and Longespicatae (Cyperaceae). Black circles, C. arctogena A; white circles, C. arctogena B; white triangles, C. capitata A; black triangles, C. capitata B; squares, C. monostachya; cross, C. oreophila; diamond, C. runssoroensis. The dark grey and the dashed regions indicate the distribution of C. arctogena and C. capitata, respectively, obtained from the World Checklist of Selected Plant Families (http://apps.kew.org/wcsp).

2 Journal of Biogeography ª 2015 John Wiley & Sons Ltd 158 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena Bipolar disjunction in Carex arctogena

DNA sequences with bioclimatic data could thus be valuable known to blur signals of migration and isolation (Schaal & in clarifying the evolutionary history of this bipolar species. Olsen, 2000). Consequently, this region was used for phylo- The goal of this study was to determine which of the four genetic purposes alone and was only amplified for a subset classic hypotheses used to account for bipolar taxa could best of samples. explain the distribution of C. arctogena. By evaluating the Nuclear and plastid regions were amplified and sequenced combined evidence provided by phylogenetic reconstructions, following the conditions described by Escudero et al. (2008) molecular dating and bioclimatic data, we will be able to test and Starr et al. (2009), respectively. Minor adjustments (e.g. biogeographical hypotheses and to improve our understand- reagent concentrations or annealing temperature) were some- ing of the historical events that promoted the formation of times necessary in order to obtain suitable amplification the bipolar disjunction seen in C. arctogena. products. Sequence data were assembled and edited using Se- quencher 4.10 (Gene Codes, Ann Arbor, MI, USA) and subsequently submitted to GenBank (Appendix S1). MATERIALS AND METHODS Sequences were automatically aligned with muscle (Edgar, 2004) and manually adjusted using Geneious 6.1.7 (Biomat- Sampling ters, Auckland, New Zealand). Carex arctogena has a circumboreal distribution, with its range limited to Patagonia in the Southern Hemisphere Genetic variation, neutrality and selection tests (Fig. 1). It is a wind-pollinated herbaceous hemicryptophyte that generally occurs in arctic–alpine habitats and wind- Nucleotide diversity (p; Nei, 1987) and haplotype diversity exposed heaths where the soil water content is low. We (Hd; Nei & Tajima, 1983) were calculated for the amplified obtained plant material representing the entire range of chloroplast regions of C. arctogena and C. capitata in DnaSP C. arctogena (55 populations), as circumscribed by Egorova 5.10 (Librado & Rozas, 2009). DnaSP was also used to test (1999). We also included 36 populations of C. capitata and for molecular selection in atpF–atpH, rps16 and matK with one population of C. oreophila. Two East African species Tajima’s D (Tajima, 1989) and Fu and Li’s D* and F* (Fu & from Carex sect. Longespicatae Kuk.,€ Carex runssoroensis K. Li, 1993) neutrality tests. Selective pressure on matK was Schum. and Carex monostachya A. Rich., were also sampled evaluated using the codon-based Z test (Nei & Gojobori, (one and two populations, respectively; Fig. 1, and see 1986). To test the null hypothesis of neutral selection, the Appendix S1 in Supporting Information), because molecular number of synonymous substitutions per synonymous site studies suggest that C. sect. Longespicatae is sister to C. sect. (dS), the number of non-synonymous substitutions per non- Capituligerae (e.g. Starr & Ford, 2009). Finally, we used Ca- synonymous site (dN), and their variances (estimated by rex obtusata Lilj., Carex rupestris All. and Uncinia triquetra bootstrap over 10,000 pseudoreplicates) were calculated for Kuk.€ as outgroups (Starr & Ford, 2009). For all species, one each pair of sequences in Mega 4 (Tamura et al., 2007). individual per population was sampled, except for five popu- Gaps or missing data were deleted in the pairwise distance lations of C. arctogena that consisted of two individuals each estimation. Because they showed incongruence due to posi- (Appendix S1). Samples used for the molecular study were tive selection, we removed the matK sequences of C. mono- obtained from silica-dried leaf material collected in the field stachya and C. runssoroensis from subsequent phylogenetic and from herbarium specimens (Appendix S1). Vouchers for analyses (see Results), mirroring the removal by Gehrke et al. new collections have been deposited in the herbaria CAN, SI (2010) of ITS sequences that showed incongruence between and UPOS. samples.

PCR amplification and sequencing Phylogenetic analyses

All regions were amplified by polymerase chain reaction We obtained a total of 19 sequences of ITS, 87 of atpF–atpH, (PCR) from total genomic DNA extracted as described by 85 of matK and 49 of rps16 (Appendix S1). The ITS region Starr et al. (2009). We amplified the nuclear ITS region was only analysed in combination with the plastid regions (using the primers ITSA and ITS4; White et al., 1990; Blatt- due to the low number of sequences obtained. The three ner, 1999) and three chloroplast DNA (cpDNA) regions: the plastid loci were analysed independently, to check for incon- atpF–atpH spacer, using primers atpF and atpH (Fazekas gruence, and in combination using maximum likelihood et al., 2008); a portion of the matK gene, using primers (ML) and Bayesian inference (BI). The combined nuclear matK 2.1f_J and matK 5r_J (Plant Working Group, Royal and plastid matrix consisted of 107 sequences with 2835 sites Botanical Gardens Kew, http://www.kew.org/barcoding/pro- (see Appendix S1). Maximum-likelihood analyses were per- tocols.html modified by Chouinard, 2010), and the rps16 formed using RAxML 7.2.6 (Stamatakis, 2006), with a intron, using primers rps16F and rps16R (Shaw et al., 2005). GTRGAMMA model of sequence evolution and node support The ITS region has been one of the most useful markers for assessed via 1000 bootstrap (BS) pseudoreplicates. Bayesian inferring plant phylogenies at low taxonomic levels, but analyses were executed in MrBayes 3.2 (Ronquist et al., concerted evolution within this multicopy gene family is 2012) using the most appropriate nucleotide substitution

Journal of Biogeography 3 ª 2015 John Wiley & Sons Ltd 159 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena T. Villaverde et al. model for each partition as chosen by jModelTest (Posada, 2001). Because the range limits of species and lineages can 2008) under the Akaike information criterion (AIC). The be influenced by spatial variation in ecological factors selected nucleotide substitution models were HKY for atpF– (Wiens, 2011), we obtained values for 19 bioclimatic vari- atpH, HKY+I for matK, GTR for rps16, HKY+I for ITS1, JC ables (Appendix S1) as described by Escudero et al. (2013) for ITS 5.8S and GTR for ITS2 (Appendix S1). The Markov for each sampled population of species in Carex sections Ca- chain Monte Carlo (MCMC) search was run for five million pituligerae and Longespicatae. To characterize the climatic generations with one tree sampled every 1000 generations niche space occupied by each species, we performed a princi- and two simultaneous analyses (‘Nruns = 2’) each of four pal components analysis (PCA) of the climatic dataset using Markov chains (‘Nchains = 4’) started from different random the ‘prcomp’ function (sdev, rotation, centre and scale trees. The first 20% of trees were discarded from each run as options were set as TRUE) and a phylogenetic PCA using burn-in. A Bayesian majority-rule consensus tree was calcu- the ‘phyl_pca’ function in the phytools package (assuming lated in MrBayes with posterior probability (PP) values as a Brownian motion and covariance matrix option; Revell, measure of clade support. Trees were edited using FigTree 2009) in R (R Development Core Team, 2014). A phyloge- 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). netic size-correction was performed in our dataset for non-independence among the observations for lineages. We represented the data associated with the most important Haplotype network bioclimatic variables retained in the PCA for C. arctogena in We obtained the genealogical relationships among all three boxplots. cpDNA haplotypes using the plastid matrix and statistical parsimony as implemented in tcs 1.21 (Clement et al., RESULTS 2000). The maximum number of differences resulting from single substitutions among haplotypes was calculated with Haplotype diversity and neutrality tests 95% confidence limits. The only informative indel (atpF– atpH region) was coded as a presence/absence character for The number of cpDNA haplotypes and haplotype diversity analysis. Gaps due to mononucleotide repeat units (poly-T were highest in matK (Nh, 6; Hd, 0.746; nucleotide diversity, and poly-A) are considered to be highly homoplastic (Kelch- p, 0.00397), whereas nucleotide diversity was highest in ner, 2000) and were therefore treated as missing data. atpF–atpH (Nh, 5; Hd, 0.725; p, 0.00442; Appendix S1). The number of segregating sites was eight in both matK and atpF–atpH, twice that in rps16. A significant departure from Divergence-time estimation neutrality was found in matK sequences (F*-test, P < 0.05; Dated phylogenies were estimated for the nuclear and plastid Appendix S1). Estimates of the average within-group nucleo- matrix in beast 1.7.5 (Drummond et al., 2012). All matK tide substitution rates for matK revealed significant positive sequences were excluded because run convergence was ham- selection (dN > dS) in C. monostachya and C. runssoroensis. pered by incongruence in matK, which showed a significant The matK sequences for these species were therefore elimi- departure from neutrality (Appendix S1). The analysed nated from the subsequent analyses as they could affect the matrix therefore consisted of 94 ITS, atpF–atpH and rps16 results of phylogenetic reconstructions. Selective pressure has sequences with an aligned length of 2089 sites. All phyloge- also been detected on matK in other plant groups (e.g. nies were estimated using an uncorrelated log-normal relaxed McNeal et al., 2009) and in other chloroplast regions (e.g. clock model. A normal age prior with a mean of 13.20 Ma Kapralov & Filatov, 2007). 2.5 Myr was applied to the crown node, based on the pre- vious estimate for the divergence of Carex sections Capituli- Phylogenetic reconstruction gerae and Longespicatae from the outgroups in the analysis of Escudero & Hipp (2013). Analyses were conducted using two Bayesian-inference (BI) and ML analyses revealed strong sup- independent MCMC runs of 40 million generations each, port (97% BS / 1.00 PP, Fig. 2) for a clade including both assuming a birth–death tree prior with a mean substitution sections. Carex monostachya was poorly supported as sister rate set at 1.0. Run convergence and burn-in were assessed to a large polytomy composed of C. runssoroensis plus C. in Tracer 1.5 (Rambaut & Drummond, 2009). Maximum- sect. Capituligerae. Carex sect. Capituligerae was retrieved as clade-credibility (MCC) trees were calculated with an unresolved group with four main lineages (see below). TreeAnnotator 1.7.2 (Drummond & Rambaut, 2007) using Neither C. arctogena nor C. capitata was resolved as a mono- a posterior probability limit of 0.9 and the mean heights phyletic taxon; instead, two different geographically defined option. lineages were detected for each species: (1) C. arctogena line- age A (90% BS / 0.71 PP) includes samples from Europe and North and South America; (2) C. arctogena lineage B (91% Climatic environment – ecological niche BS / 0.90 PP) only includes samples from western North Carex arctogena and C. capitata are known to have different America; (3) C. capitata lineage A (88% BS / 0.78 PP) ecological preferences in Scandinavia (Reinhammar & Bele, includes samples from Russia; and (4) C. capitata lineage

4 Journal of Biogeography ª 2015 John Wiley & Sons Ltd 160 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena Bipolar disjunction in Carex arctogena

Uncinia triquetra ARG 1 C. obtusata WYO 1 C. obtusata WYO 2 Outgroups 98 C. obtusata WYO 3 C. rupestris SPA C. monostachya KEN 1 0.96 C. monostachya KEN 2 C. runssoroensis KEN 69 C. oreophila TUR C. capitata MAG 1 0.78 C. capitata MAG 2 Russia 88 C. capitata MAG 3 C. capitata A C. capitata YAK 4 94 C. arctogena ARG 1 C. arctogena ARG 2 C. arctogena ARG 3 C. arctogena ARG 4 C. arctogena ARG 5 1 C. arctogena ARG 6 C. arctogena ARG 7 97 C. arctogena ARG 8 C. arctogena ARG 9 C. arctogena ARG 10 C. arctogena MAN 11 0.71 C. arctogena MAN 12 C. arctogena A C. arctogena MAN 13 90 C. arctogena BRC 14 Northern & C. arctogena QUE 15 C. arctogena QUE 16 C. arctogena QUE 17 Southern Sect. C. arctogena LAB 18 0.73 C. arctogena SAS 19 C. arctogena ONT 20 Hemispheres Capituligerae C. arctogena ONT 21 C. arctogena NWH 22 + Longespicatae C. arctogena GNL 23 C. arctogena GNL 24 C. arctogena FIN 25 90 87 C. arctogena QUE 26 C. capitata MAG 5 C. capitata RUN 6 C. capitata FIN 7 C. capitata FIN 8 C. capitata FIN 9 C. capitata SWE 10 C. capitata SWE 11 C. capitata SWE 12 C. capitata SWE 13 C. capitata NOR 14 C. capitata ICE 15 C. capitata ICE 16 C. capitata ICE 17 C. capitata ICE 18 0.92 C. capitata ASK 23 C. capitata BRC 21 97 C. capitata BRC 22 C. capitata B C. capitata ALB 19 C. capitata MAN 23 C. capitata MAN 24 Northern C. capitata MAN 25 C. capitata MAN 26 C. capitata MAN 27 Hemisphere C. capitata MAN 28 C. capitata MAN 29 C. capitata ONT 30 C. capitata ONT 31 C. capitata YUK 32 C. capitata YUK 33 C. capitata YUK 34 C. capitata NWT 35 C. capitata NWT 36 60 C. capitata SAS 37 C. arctogena ORE 27 C. arctogena ORE 28 C. arctogena WYO 29 C. arctogena ALB 30 C. arctogena WAS 31 C. arctogena WAS 32 C. arctogena MNT 33 C. arctogena MNT 34 C. arctogena MNT 35 C. arctogena UTA 36 C. arctogena UTA 37 C. arctogena NEV 38 C. arctogena COL 39 C. arctogena COL 40 C. arctogena COL 41 0.9 C. arctogena COL 42 C. arctogena COL 43 91 C. arctogena COL 44 C. arctogena CAL 45 C. arctogena B C. arctogena CAL 46 C. arctogena CAL 47 W North America C. arctogena CAL 48 C. arctogena CAL 49 C. arctogena CAL 50 C. arctogena CAL 51 C. arctogena CAL 52 C. arctogena CAL 53 C. arctogena CAL 54 C. arctogena CAL 55 C. arctogena CAL 56 C. arctogena CAL 57 C. arctogena CAL 58 C. arctogena CAL 59 87 C. arctogena CAL 60 C. arctogena CAL 61 0.003

Figure 2 Majority-rule (50%) consensus tree from the Bayesian analysis of nuclear and chloroplast sequences from Carex sections Capituligerae and Longespicatae (Cyperaceae). Uncinia triquetra, Carex rupestris and C. obtusata were used as outgroups. Numbers above branches represent Bayesian posterior probabilities (> 0.7 PP); numbers below branches represent bootstrap values (> 60% BS) from the maximum-likelihood analyses. The grey rectangle highlights C. arctogena samples from the Southern Hemisphere. Abbreviations after names correspond to geographical regions of the world (Brummitt, 2001) followed by population number.

Journal of Biogeography 5 ª 2015 John Wiley & Sons Ltd 161 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena T. Villaverde et al.

B (97 BS / 0.92 PP) comprises samples from North America, 0.19–1.66 Ma, Table 1). The grouping of Carex arctogena A Europe and Russia. with C. runssoroensis did not receive statistical support in the MCC tree above 0.9. Haplotype network Climatic environment The cpDNA haplotype network (Fig. 3) revealed 10 haplo- types and five missing haplotypes. Geographical structure The PCA of the climatic dataset from 94 total populations was detected in most lineages, similar to that found in the consisting of C. arctogena (53 individuals; two populations phylogenetic reconstruction. We found one unique haplotype had missing data in the WorldClim database), C. capitata in C. arctogena lineage A, four in C. arctogena lineage B, one (35), C. oreophila (3), C. monostachya (2) and C. runssoroen- in C. capitata lineage A and two in C. capitata lineage B. sis (1) showed that principal component 1 (PC1) explained There is a haplotype shared by eight samples of C. arctogena 98.99% of variance and PC2 explained 0.78% (Fig. 5). The A, C. oreophila, C. monostachya and one individual of variables with the highest loadings on PC1 were temperature C. capitata B. Carex runssoroensis occupied a central position seasonality (BIO4), the mean temperature of the coldest in the network. The 10 C. arctogena samples from the South- quarter (BIO11), the minimum temperature of the warmest ern Hemisphere shared the same haplotype as the 10 North- month (BIO6) and isothermality (BIO3; Appendix S1). Max- ern Hemisphere samples of C. arctogena A. ima and minima for each lineage are shown in Table 2. Sim- ilar results were obtained when the analysis was not corrected for the phylogeny (results not shown). Northern Estimation of divergence times and Southern Hemisphere samples of C. arctogena A were The dating analyses produced a partly incongruent topology clearly separated into two groups. The boxplots of the vari- with respect to the BI and ML analyses presented above ables with the highest loadings revealed that northern popu- (Fig. 4, Table 1). The divergence time of the clade compris- lations of C. arctogena A tolerate greater temperature ing Carex sections Capituligerae and Longespicatae was oscillations through the year and a wider range of minimum 6.76 Ma (95% highest posterior density interval, HPD, 3.05– temperatures during the coldest month than populations 11.29 Ma), which falls in the late Miocene to early Pliocene. from the Southern Hemisphere (Table 2, Fig. 6a,b). The diversification of the clade consisting of C. arctogena, C. capitata, C. oreophila and C. runssoroensis is estimated to DISCUSSION have begun 5.0 Ma (95% HPD 2.10–8.03 Ma). The crown nodes of the main lineages obtained in the phylogeny Origin of the bipolar distribution of C. arctogena were placed in the late Pleistocene (C. monostachya: 0.13 Ma, 95% HPD 0–0.51 Ma; C. capitata A plus C. oreophila: Our study provides strong evidence for a recent origin of the 0.37 Ma, 95% HPD 0.01–1.17 Ma; C. capitata B: 0.68 Ma, bipolar disjunction in C. arctogena lineage A. The divergence 95% HPD 0.14–1.39 Ma; C. arctogena B: 0.81 Ma, 95% HPD time for the clade comprising Carex sections Capituligerae

C. C. arctogena arctogena B C. B (3) (13) arctogena B C. (12) arctogena B W North America (1)

North America & C. C. Eurasia capitata runssoroensis (1) A (4) C. Figure 3 tcs haplotype network of arctogena A concatenated cpDNA sequences of Carex C. (20) C. monostachya sections Capituligerae and Longespicatae capitata (2) B (Cyperaceae). Circles represent haplotypes, (4) C. oreophila (1) North & South lines represent single mutational steps and C. arctogena A America small black circles are missing haplotypes. C. (8) Circle shades indicate species, and numbers C. capitata B capitata (1) in parentheses indicate the number of B samples per haplotype. Shaded and dashed (22) Eurasia, North squares represent the geographical America & Africa distributions of lineages.

6 Journal of Biogeography ª 2015 John Wiley & Sons Ltd 162 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena Bipolar disjunction in Carex arctogena

3.84 Outgroups

0.13 C. monostachya Kenya

6.76 0.37 C. capitata A (Russia) + C. oreophila Turkey

Sect. 5.00 Capituligerae C. arctogena A (North & 2.18* South America) + + C. runssoroensis Kenya Longespicatae 0.68 C. capitata B (North America & Eurasia)

0.81 C. arctogena B (W North America)

MIOCENE PLIOCENE PLEISTOCENE

15.0 12.5 10.0 7.5 5.0 2.5 0.0Ma

Figure 4 Maximum-credibility-clade phylogeny from the Bayesian divergence-time analysis of Carex sections Capituligerae and Longespicatae (Cyperaceae) using a combined matrix of ITS and atpF–atpH and rps16. Node bars represent the 95% highest posterior density intervals for the divergence-time estimates of nodes with posterior probabilities above 0.9. See Table 1 for posterior probabilities and ages inferred for clades. The asterisk denotes that the C. arctogena and C. runssoroensis clade has a posterior probability below 0.5.

Table 1 Divergence dates of clades resolved in Carex sections Capituligerae and Longespicate (Cyperaceae), presented as posterior probabilities, mean and median time to the most common recent ancestor in millions of years (Ma) and 95% highest posterior density (HPD) interval obtained from the divergence time analyses of the combined nuclear (ITS) and plastid (atpF–atpH and rps16) matrix.

Clade Posterior probability Mean (Ma) Median (Ma) 95% HPD interval

Carex sect. Capituligerae + Longespicatae 1.00 6.76 6.44 3.05 11.29 C. monostachya 0.96 0.13 0.05 0.00 0.51 C. runssoroensis + C. oreophila 0.64 5.00 4.81 2.31 8.03 + C. arctogena + C. capitata C. oreophila + C. capitata A 0.61 0.37 0.22 0.01 1.17 C. capitata B 0.51 0.68 0.60 0.14 1.39 C. arctogena B 0.96 0.81 0.72 0.19 1.66

and Longespicatae (crown node: 6.76 Ma, 95% HPD 3.05– clade (Fig. 2) and our haplotype data demonstrate that pop- 11.29 Ma; Fig. 4, Table 1) is far younger than the trans-trop- ulations from both hemispheres share identical cpDNA hapl- ical highland bridges (c. 195 Ma; Scotese et al., 1988) and we otypes over the 2207 bp of three chloroplast markers therefore reject the vicariance hypothesis for the bipolar dis- (Fig. 3). This clearly suggests that C. arctogena A is a bipolar junction of C. arctogena (Du Rietz, 1940). If convergent evo- monophyletic clade, so we reject a hypothesis of convergent lution could explain the bipolar distribution of C. arctogena, evolution (Stern, 2013). northern and southern populations of the species would not The bipolar disjunction is best explained by long-distance share an immediate common ancestor. In contrast, our phy- dispersal, which may have been either by mountain-hopping logenetic results place all C. arctogena A samples in a single (‘stepping stones’) or by a direct event (a ‘giant leap’). This

Journal of Biogeography 7 ª 2015 John Wiley & Sons Ltd 163 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena T. Villaverde et al.

C. arctogena A NH A SH 4 C. arctogena C. arctogena B C. capitata A C. capitata B C. monostachya C. oreophila C. runssoroensis PC2

Figure 5 Scatter plot of the first two principal components, which explain 99.78%, from the principal components -8 -6 -4 -2 0 2 analysis depicting the position of the -2 0 2 4 6 8 10 samples of Carex sections Capituligerae and Longespicatae (Cyperaceae) in climatic niche PC1 space.

Table 2 Maximum and minimum values for climatic variables ecophysiological adaptations to crossing the short-day condi- with the highest loadings on principal component 1 by groups tions of the tropical alpine environment seem to be necessary of Carex arctogena and C. capitata: BIO4, temperature (Heide, 2002), but we are not aware of any published fossil seasonality; BIO6, minimum temperature of the coldest month; records or any other evidence for the occurrence of C. arcto- BIO11, mean temperature of the coldest quarter; BIO3, gena in areas between northern North America and southern isothermality. South America. If C. arctogena had migrated to South Amer- BIO4 BIO6 BIO11 BIO3 ica by the slow and gradual means predicted by mountain- hopping, we would expect that such a process would leave a C. arctogena A min. 31.15 34.8 27.9 17% max. 150.98 1.7 4.8 51% trace of genetic differences in the plastid loci of populations C. arctogena B min. 51.78 20.5 12.9 31% from both hemispheres (Brochmann et al., 2003; Scotland, max. 96.09 0.6 7.2 46% 2011), as has been shown for other bipolar species (Vollan C. capitata A min. 153.45 47.1 42.0 16% et al., 2006; Escudero et al., 2010). Although we cannot com- max. 205.98 34.8 29.2 20% pletely reject the mountain-hopping hypothesis, the absence C. capitata B min. 37.44 33.9 29.0 16% of genetic variability between populations of C. arctogena A max. 148.76 4.9 1.7 36% from both hemispheres fits better with a recent and direct C. arctogena A Northern min. 64.38 34.8 27.9 17% Hemisphere max. 150.98 13.9 10.3 27% long-distance dispersal. Direct long-distance dispersal has C. arctogena A Southern min. 31.15 5.1 0.1 45% been shown to be remarkably frequent in some other species Hemisphere max. 51.98 1.7 4.8 51% of Cyperaceae (e.g. Viljoen et al., 2013). The utricle surrounding Carex fruit can show some features for wind-dispersal, as seen in Carex physodes (Egorova, 1999) could have occurred during some of the last cold periods at or for animal-dispersal as seen in Carex microglochin (Savile, the end of the Pliocene or in the Pleistocene, which 1972). However, with the exception of the bladder-like utricle, expanded the polar regions in both hemispheres (Raven, fruits and surrounding fruit structures of Carex generally lack 1963; Ball, 1990), or even at present times. Given that all any obvious morphological features for dispersal by abiotic or other taxa in Carex section Capituligerae and all but one biotic forces. The perigynia of Carex arctogena do not have any haplotype are found in the Northern Hemisphere, our data apparent mechanism for dispersal; even the aculeolate teeth on suggest that this dispersal occurred from the Northern to the the margin of the perigynia are variable in number, sometimes Southern Hemisphere. being entirely absent. We suggest that relatively unspecialized The remaining question is: which mechanism better structures for dispersal might play a role in the distribution of explains the bipolar disjunction – mountain-hopping or C. arctogena. We regard the hypothesis of non-standard vec- direct long-distance dispersal? The mountain-hopping tor-mediated dispersal, either by abiotic or biotic forces, as a hypothesis (Ball, 1990) proposes a stepwise long-distance possible explanation of the bipolar disjunction of C. arctogena. migration by mountain peaks as stepping-stones for polar It is possible that populations of C. arctogena in the South- and temperate taxa to cross the ecological barrier presented ern Hemisphere may have been the result of an accidental by the tropics. A route connecting North and South America anthropogenic introduction. In this scenario, adaptation to through the American Cordillera has been in place since local environmental conditions, biotic interactions and the late Miocene (Smith, 1986). For species of Carex,no demographic processes of this species would all have been

8 Journal of Biogeography ª 2015 John Wiley & Sons Ltd 164 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena Bipolar disjunction in Carex arctogena

(a) (b)

Figure 6 Carex arctogena boxplots comparing the four bioclimatic variables with the highest loadings on the first component of the bioclimatic PCA for the different lineages found (A and B) and for Northern and Southern Hemisphere samples (A NH and A SH, respectively). (a) Temperature seasonality (BIO4); (b) (c) (d) minimum temperature of the warmest month (BIO6); (c) mean temperature of the coldest month (BIO11); (d) isothermality (BIO3). Each box represents the interquartile range which contains 50% of the values and the median (horizontal line across the box). The whiskers extending from the box represent the highest and lowest values observed, excluding outliers (dots). established relatively quickly (Theoharides & Dukes, 2007). Their breeding ranges closely match the current distribution Populations of C. arctogena in Patagonia occur in well-con- of C. arctogena A in both hemispheres (Fig. 1). Although served habitats and most are only accessible on foot. Speci- current bird migratory patterns do not necessarily coincide mens of C. arctogena from Patagonia are few in the South with past migrations, these observations suggest that the American herbaria BA, BAA, BAB, BCRU, HIP and SI, with bipolar disjunction in C. arctogena may have originated via some dating to the late 1880s, when the human influence in bird-mediated long-distance dispersal. Additionally, dispersal the southernmost parts of South America was very limited. may occur through accidental displacement – vagrant birds Although we cannot rule out an anthropogenic introduction or migrants, such as those flying to Australia or New Zea- of this species to South America, it seems unlikely. land, deviating widely from their normal route (Battley et al., Bird-mediated direct long-distance dispersal from North 2012). With satellite telemetry, Gill et al. (2009) recorded America has already been used to explain a bipolar disjunc- transoceanic flights of bar-tailed godwits (Limosa lapponica tion in crowberries (Empetrum; Popp et al., 2011). Most baueri) from Alaska to New Zealand and showed that they migratory birds that disperse seeds live in temperate and can fly 10,153 km ( 1043 SD) non-stop in 7.8 days ( 1.3 boreal regions (Wheelwright, 1988). For birds to act as vec- SD). This extraordinary flight, combined with species that tors for seed dispersal by endo- or ectozoochory, the seeds can be preferentially chosen for fuel, could help species such must have morphological features for association with these as C. arctogena to achieve a bipolar distribution by means of animals, and must be able to maintain their viability after direct long-distance dispersal. intestinal transit to allow for establishment in new environ- ments (Gillespie et al., 2012). Although Carex arctogena Climatic regime differentiation fruits lack obvious morphological features for zoochorous dispersal, other structures or features that are not directly Theoretically, C. arctogena A is most likely to become estab- related with dispersal syndromes may be involved, including lished at the high latitudes and elevations in the Southern anatomical features such as deposits of silica in the pericarp Hemisphere that have similar climatic conditions to those of that harden seeds (Graven et al., 1996; Prychid et al., 2004). northern populations (Carlquist, 1966). Although our results These silica deposits could protect seeds when passing from the bioclimatic data show that Southern Hemisphere through birds’ alimentary tracts (Graven et al., 1996) but populations currently differ from Northern Hemisphere pop- could also make the seeds as hard as pebbles and useful for ulations of C. arctogena A in their climatic niches (Fig. 6), grinding other organic material in bird gizzards. Carex fruits differences in community assembly, which suggest differences could therefore be doubly preferred by birds – both as nour- in competitive interactions, may explain how C. arctogena A ishment and as gastroliths (Alexander et al., 1996). was able to establish itself in South America after one or Some birds from North America, such as the pectoral more initial dispersal events (Waters, 2011). Such differences sandpiper, Calidris melanotos (Holmes & Pitelka, 1998), and could have allowed C. arctogena to shift into new habitats the lesser yellowlegs, Tringa flavipes (Tibbitts & Moskoff, and climate zones (Broennimann et al., 2007). Alternatively, 1999), are known to feed in sedge meadows before migrating establishment could have taken place at a time when both southwards to their wintering grounds in South America. areas had similar climatic conditions.

Journal of Biogeography 9 ª 2015 John Wiley & Sons Ltd 165 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena T. Villaverde et al.

CONCLUSIONS B.D., Schuckard, R., Melville, D.S. & Riegen, A.C. (2012) Contrasting extreme long-distance migration patterns in Evidence from multiple analytical approaches was used to bar-tailed godwits Limosa lapponica. Journal of Avian Biol- infer the possible mechanisms underlying the distribution of ogy, 43,21–32. a bipolar species. Bioclimatic data, phylogenetic and phylo- Blattner, F.R. (1999) Direct amplification of the entire ITS geographical analyses and divergence-time estimates have region from poorly preserved plant material using recom- been integrated to test hypotheses that are traditionally used binant PCR. BioTechniques, 27, 1180–1186. to account for the origin of bipolar distributions at the spe- Brochmann, C., Gabrielsen, T.M., Nordal, I., Landvik, J.Y. & cies level. Our study highlights the importance of long-dis- Elven, R. (2003) Glacial survival or tabula rasa? The history tance dispersal in explaining this extraordinary pattern of of North Atlantic biota revisited. Taxon, 52, 417–450. plant distribution, although further comparative studies Broennimann, O., Treier, U.A., Muller-Sch€ €arer, H., Thuiller, using multiple bipolar species are necessary to test the same W., Peterson, A.T. & Guisan, A. (2007) Evidence of cli- explanation in other phylogenetically independent cases. matic niche shift during biological invasion. Ecology Let- ters, 10, 701–709. ACKNOWLEDGEMENTS Brummitt, R.K. (2001) World geographical scheme for record- ing plant distributions, 2nd edn. Hunt Institute for Botani- We thank the staff of the following herbaria for giving us cal Documentation, Pittsburgh, PA. access to their collections and providing plant material: A, Carlquist, S. (1966) The biota of long-distance dispersal. I. ALA, BA, BAA, BAB, BCRU, BRY, C, CAN, CAS, CCO, Principles of dispersal and evolution. Quarterly Review of CHSC, COLO, DAO, E, GH, H, HIP, ICEL, M, MICH, Biology, 41, 247–270. MONTU, MOR, O, OSC, RM, RMS, SI, UBC, UNM, UPOS, Chouinard, B.N. (2010) DNA barcodes for the Cariceae (Ca- UTEP, WIN and WTU. Thanks are also due to three anon- rex & Kobresia, Cyperaceae) of North America, north of ymous referees and to the editor Robert Whittaker. We Mexico. MSc Thesis, University of Ottawa, Ottawa, ON. would also like to thank E. Maguilla (Universidad Pablo de Clement, M., Posada, D. & Crandall, K.A. (2000) TCS: a Olavide, Seville, Spain; UPO) for his help with map editing, computer program to estimate gene genealogies. Molecular M. Gosselin (Canadian Museum of Nature, Ottawa, Canada; Ecology, 9, 1657–1660. CMN) for providing information related to bird dispersal, Crisp, M.D., Trewick, S.A. & Cook, L.G. (2011) Hypothesis W. Sawtell (University of Ottawa, Canada) and P. Vargas testing in biogeography. Trends in Ecology and Evolution, (Real Jardın Botanico de Madrid, Spain) for assistance in 26,66–72. plant collections, and R. Bull (CMN), M. Mıguez and F. J. Darwin, C. (1859) On the origin of species by means of natu- Fernandez (UPO) for technical support. In addition, we are ral selection. John Murray, London. grateful to University of Ottawa undergraduate students A. Drummond, A.J. & Rambaut, A. (2007) BEAST: Bayesian Ginter for translations of Russian label data and J. E. Pender evolutionary analysis by sampling trees. BMC Evolutionary for assistance with databasing label data and DNA sequenc- Biology, 7, 214. ing. This research was supported by a Natural Sciences and Drummond, A.J., Suchard, M.A., Xie, D. & Rambaut, A. Engineering Research Council of Canada (NSERC) Discovery (2012) Bayesian phylogenetics with BEAUti and the BEAST Grant to J.R.S. and by a Talentia Scholarship from the 1.7. Molecular Biology and Evolution, 29, 1969–1973. Regional Ministry of Economy, Innovation, Science and Du Rietz, G.E. (1940) Problems of the bipolar plant distribu- Employment of Andalusia awarded to T.V. for MSc research tion. Acta Phytogeographica Suecica, 13, 215–282. at the University of Ottawa. Further support was provided Edgar, R.C. (2004) MUSCLE: multiple sequence alignment by the Spanish Ministry of Science and Technology through with high accuracy and high throughput. Nucleic Acids project CGL2012-38744 and from the Regional Ministry of Research, 32, 1792–1797. Economy, Innovation, Science and Employment of Andalucia Egorova, T.V. (1999) The sedges (Carex L.) of Russia and through the project RNM-2763. adjacent states (within the limits of the former USSR). Mis- souri Botanical Garden Press, St Louis, MO. Escudero, M. & Hipp, A. (2013) Shifts in diversification rates REFERENCES and clade ages explain species richness in higher-level Alexander, S.A., Hobson, K.A., Gratto-Trevor, C.L. & Dia- sedge taxa (Cyperaceae). 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12 Journal of Biogeography ª 2015 John Wiley & Sons Ltd 168 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Journal of Biogeography

SUPPORTING INFORMATION

Direct long-distance dispersal best explains the bipolar distribution of Carex arctogena (Carex sect. Capituligerae, Cyperaceae)

Tamara Villaverde, Marcial Escudero, Santiago Martín-Bravo, Leo P. Bruederle, Modesto Luceño and Julian R. Starr

Appendix S1 Studied material, molecular characteristics of the amplified regions and results from the principal components analysis of 19 bioclimatic variables from the WorldClim database, uncorrected and corrected for phylogeny.

Table S1 List of material studied. Table S2 Characteristics of the DNA regions sequenced. Table S3 Locus information for the regions amplified in the study. Table S4 Average within-group nucleotide substitution estimates for the matK gene of the complete dataset. Table S5 Loadings matrix obtained by the principal components analysis not corrected by phylogeny of 19 bioclimatic variables on Carex sections Capituligerae and Longespicatae. Table S6 Loadings matrix obtained by the principal components analysis corrected by phylogeny of 19 bioclimatic variables on Carex sections Capituligerae and Longespicatae. Table S7 Bioclimatic variables used.

169 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Table S1 List of material studied of Carex arctogena, C. capitata, C. monostachya, C. oreophila, C. runssoroensis, C. rupestris, C. obtusata and Uncinia triquetra including population code, coordinates, voucher information, corresponding clade and GenBank accessions for markers used for molecular studies. Population codes correspond to geographical regions of the world (Brummitt, 2001) and population number.

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. arctogena BRC 55.61667 ΫͳʹʹǤ͸ͷͲͲͲ Canada, British Columbia, Pine Pass. G. arctogena A — KP996286 KP996374 — 14 W. Argus 8831. 12/7/1973. (CAN- NH 372267) C. arctogena FIN 68.22200 23.62700 Finland, Enontekiö. H. Väre 17177. arctogena A KP984469 — — — 25 17/7/2006. (H-809948) NH C. arctogena GNL 67.90000 ΫͶͻǤͶͳ͸͸͹ Greenland, Arfersoprflk. B. Fredskild arctogena A — KP996285 KP996372 — 23 & V. Dalgaard s.n. 19/8/1987. (COLO- NH 456814) C. arctogena GNL 64.05000 ΫͶͻǤͻͷͲͲͲ Greenland, Pingorssuaq kitdleq. S. arctogena A KP984471 — — — 24 Hanfgam 83-175. 11/7/1983. (C- NH 17/2009N3) C. arctogena LAB 51.88333 Ϋ͸ͷǤͻͷͲͲͲ Canada, Labrador, Esker area. Y. arctogena A — KP996287 KP996375 KP996451 18 Mäkinen & E. Kankainen s.n. NH 21/7/1967. (CAN-314758) C. arctogena MAN 60.00000 Ϋͻ͸Ǥͺ͵͵͵͵ Canada, Manitoba, Baralzon Lake. H. J. arctogena A — KP996284 KP996371 — 11 Scoggan 22434 & W. K. W. Baldwin. NH 18/7/1950. (WIN, 22434) C. arctogena MAN 58.06000 Ϋ͸ͺǤ͵ͻͻͲͲ Canada, Quebéc, Fort Chimo. A. arctogena A — KP996281 KP996368 — 12 Legault 6782. 22/7/1963. (COLO- NH 491481)

170 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. arctogena MAN 59.36667 Ϋͻ6.23333 Canada, Manitoba, Hudsons Bay Co., arctogena A — KP996289 KP996377 KP996453 13 Duck Lake. H. J. Scoggan 8288 & W. K. NH W. Baldwin. 19/8/1950. (CAN- 201506) C. arctogena ONT 54.36280 ΫͺͶǤͶ͹ͻͷͲ Canada, Ontario, Kenora District, arctogena A — KP996354 — — 20 Patricia Portion. J. L. Riley 11856. NH 12/8/1980. (CAN-462937) C. arctogena ONT 55.11160 Ϋͻ͵Ǥ͵ͷͷͻͲ Canada, Ontario, Hudson Bay arctogena A — KP996282 KP996369 — 21 Lowlands. A. E. Porsild et al. 19898. NH 4/7/1957. (CAN-278707) C. arctogena QUE 59.26667 Ϋ͹ʹǤͷͺ͵͵͵ Canada, Quebec, Lac Payne. A. Legault arctogena A — KP996280 KP996367 — 15 A7849. 2/8/1965. (CCO-23398) NH C. arctogena QUE 52.87400 ΫͺʹǤͺ͵͹ͲͲ Canada, Quebec, Boatswain Bay. W. K. arctogena A — KP996283 KP996370 — 16 W. Baldwin 406 et al. 8/7/1947. (CAN- NH 17333) C. arctogena QUE 52.75000 Ϋ͹͵Ǥͺͺ͵͵͵ Canada, Quebéc, Lac Jaucourt Region, arctogena A — KP996288 KP996376 KP996452 17 Lichteneger Lake. G. W. Argus 9221. NH 16/7/1974. (CAN-3779977) C. arctogena QUE 58.15000 Ϋ͸ͺǤͶͳͲͲͲ Canada, Quebec, Fort Chimo. T. Soren- arctogena A KP984474 — — — 26 sen 293. 17/7/1959. (C15/2009N4) NH C. arctogena SAS 59.91667 ΫͳͲͳǤ͸͸͸͸͹ Canada, Saskatchewan, vicinity of arctogena A — KP996290 KP996378 KP996454 19 Patterson Lake. G. W. Argus s.n. NH 20/7/1963. (CAN-282691) C. arctogena ARG ΫͷͶǤ͵͵͵ʹͲ Ϋ͸͹ǤͶͶͻ͸Ͳ Argentina, Tierra del Fuego, Tolhuin. arctogena A KP984465 — KP996361 KP996445 1 S. Martín-Bravo et al. 40SMB10(1). SH 14/1/2010. (UPOS-4271)

171 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. arctogena ARG ΫͷͶǤ͵ͷͳ͸Ͳ Ϋ͸͹Ǥ͸ͷͲͲͲ Argentina, Tierra del Fuego, Tolhuin. arctogena A KP984466 — KP996362 KP996446 2 S. Martín-Bravo et al. 35SMB10(1). SH 12/1/2010. (UPOS-4272) C. arctogena ARG Ϋͷ͵Ǥͻ͵ͲͲͲ Ϋ͸ͺǤͲͺͺͲͲ Argentina, Tierra del Fuego, Río arctogena A — KP996276 KP996362 KP996447 3 Grande. J. Starr 10015 & T. Villaverde. SH 13/1/2010. (CAN) C. arctogena ARG ΫͶͺǤ͹ͳͲͷ͸ Ϋ͹ͳǤͲͷͲͺͳ Argentina, Santa Cruz, Los Glaciares arctogena A — KP996277 KP996364 KP996448 4 National Park. J. Starr 10020 & T. SH Villaverde. 21/1/2010. (CAN) C. arctogena ARG ΫͶͺǤ͹ͳͲͷ͸ Ϋ͹ͳǤͲͷͲ81 Argentina, Santa Cruz, Los Glaciares arctogena A — KP996278 KP996365 KP996449 5 National Park. J. Starr 10020 & T. SH Villaverde. 21/1/2010. (CAN) C. arctogena ARG ΫͶͺǤͺʹͳ͵͸ Ϋ͹ͳǤͲͷʹͷͲ Argentina, Santa Cruz, Los Glaciares arctogena A — KP996279 KP996366 KP996450 6 National Park. J. Starr 10023 & T. SH Villaverde. 22/1/2010. (CAN) C. arctogena ARG Ϋͷ͵Ǥͻ͵ʹ͵Ͳ Ϋ͸ͺǤͲͺͺ͹Ͳ Argentina, Tierra del Fuego, Río arctogena A — KP996348 — KP996483 7 Grande. J. Starr 10015 & T. Villaverde. SH 13/1/2010. (CAN) C. arctogena ARG ΫͶͺǤͺʹͳ͵͸ Ϋ͹ͳǤͲͷʹͷͲ Argentina, Santa Cruz, Los Glaciares arctogena A — KP996349 — KP996484 8 National Park. J. Starr 10023 & T. SH Villaverde. 22/1/2010. (CAN) C. arctogena ARG ΫͶͻǤʹ͸͸͸͹ Ϋ͹ͳǤ͸͸͸͸͹ Argentina, Santa Cruz, Sierra Baguales, arctogena A — KP996350 — KP996485 9 M. K. Arroyo 85201. 16/1/1985. (HIP- SH 10500) C. arctogena ARG Ϋ͵ͷǤʹͺ͵͵͵ Ϋ͸ͻǤͷ͵͵͵͵ Argentina, Neuquén, Chos Malal. O. arctogena A — KP996353 — — 10 Boeckle et al. s.n. 30/1/1964. (BAA- SH 11368)

172 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. arctogena CAL 41.24700 ΫͳʹͳǤ͹ͷ500 USA, California, Siskiyou Co. J. D. arctogena B KP984467 — — KP996482 36 Jokerst 1823. 5/7/1983. (CHICO- 38999) C. arctogena ALB 51.61000 ΫͳͳͷǤͺʹͳͲͲ Canada, Alberta, Snow Creek Pass, A. arctogena B — KP996347 KP996436 KP996481 30 E. Porsild 22673. 29/7/1960. (CAN- 266077) C. arctogena CAL 40.10253 ΫͳʹͲǤͶͻͻͻʹ USA, California, Butte Co., J. Starr 10S- arctogena B — KP996323 KP996412 KP996465 45 054 & T. Villaverde. 6/8/2010. (CAN) C. arctogena CAL 40.10222 ΫͳʹͲǤͶͻͻͳ͹ USA, California, Butte Co., J. Starr arctogena B — KP996319 KP996408 KP996462 46 06018 & J. Thibeault. 3/8/2006. (CAN) C. arctogena CAL 40.12739 ΫͳʹͲǤͷͲͻͲ͵ USA, California, Tehama Co., Yellow arctogena B — KP996320 KP996409 KP996463 47 Pine Forest. L. Ahart 94326. 19/7/2006. (CHSC-94326) C. arctogena CAL 39.47800 ΫͳʹͲǤʹͻʹͲͲ USA, California, Sierra Co., Anderson arctogena B — KP996333 KP996422 — 48 Mdw. R. K. Gierischerisch 3493 & D. Esplin. 25/6/1969. (COLO-246761) C. arctogena CAL 40.10400 ΫͳʹͳǤͷͲʹͲͲ USA, California, Butte Co. J. Starr 10S- arctogena B — KP996322 KP996411 KP996464 49 054 & T. Villaverde. 6/8/2010. (CAN) C. arctogena CAL 39.50191 ΫͳʹͲǤͳͺ͵ʹͷ USA, California, Sierra Nevada Co. W.A. arctogena B — KP996343 KP996432 — 51 Nisbet 45. 20/7/1957. (CAN-272091) C. arctogena CAL 39.49500 ΫͳʹͲǤͶͳͳͲͲ USA, California, Sierra Co. H. Oswald arctogena B — KP996329 KP996418 KP996468 52 8221 & L. Ahart. 19/8/1996. (CHSC- 66824) C. arctogena CAL 39.42472 ΫͳʹͲǤʹͷ͸͸͹ USA, California, Nevada Co. R. Naczi arctogena B KP984464 — KP996359 — 53 11420. 3/8/2006. (US-3534689) C. arctogena CAL 39.41917 ΫͳͳͻǤ͹Ͷ͵͵͵ USA, California, Nevada Co. R. Naczi arctogena B — — KP996358 KP996443 54 11420. 3/8/2006. (CHICO-99406)

173 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. arctogena CAL 40.07700 ΫͳʹͳǤͷͷͲͲͲ USA, California, Butte Co. L. P. Janeway arctogena B KP984463 — KP996357 KP996442 55 3111. 29/7/1988. (CHICO-44118) C. arctogena CAL 38.79167 ΫͳͳͺǤͲͶͳ͸͹ USA, California, El Dorado Co. J. Pykäla arctogena B KP984468 — — — 57 et al. s.n. 23/7/1987. (H-15/2009N6) C. arctogena CAL 36.65800 ΫͳͳͺǤͶ͹͵ͲͲ USA, California, Tulare Co. S. Brush & J. arctogena B — — KP996360 KP996444 58 Oliphant 155. 24/8/1991. (CAS- 857890) C. arctogena CAL 37.92100 ΫͳͳͻǤʹͳͶͲͲ USA, California, Mono Co., Mt. Dana arctogena B — KP996326 KP996415 — 59 Plateau. D. Taylor 7550. 25/7/1979. (COLO-330874) C. arctogena CAL 37.92200 ΫͳͳͻǤʹͳͶͲͲ USA, California, Mono Co., Mt. Dana arctogena B — KP996327 KP996416 — 60 Plateau. D. Taylor 7550, 25/7/1979. (COLO-330874) C. arctogena CAL 34.49900 ΫͳͳͺǤʹͶͺͲͲ USA, California, Tulare Co., Sierra arctogena B — KP996328 KP996417 — 61 Nevada. J. T. Howell s.n. 5/8/1949. (DAO-257423) C. arctogena COL 39.83400 ΫͳͲͷǤ͸͹ͺͲͲ USA, Colorado, Clear Creek Co. W. A. arctogena B — KP996346 KP996435 KP996480 39 Weber et al. s.n. 8/8/1972. (CAN- 374041) C. arctogena COL 39.80700 ΫͳͲͷǤ͹ͳͲͲͲ USA, Colorado, Clear Creek Co., Bill arctogena B — KP996330 KP996419 KP996469 40 Moore Lake. N. Lederer s.n. 31/8/1993. (COLO-00263731) C. arctogena COL 38.02547 ΫͳͲ͸Ǥ͹ͷͻͻʹ USA, Colorado, Hinsdale Co., Gunnison arctogena B — KP996338 KP996427 KP996475 41 National Forest. J. Starr 10S-033 & T. Villaverde. 26/7/2010. (CAN) C. arctogena COL 38.02547 ΫͳͲ͸Ǥ͹ͷͻͻʹ USA, Colorado, Hinsdale Co., Gunnison arctogena B — KP996337 KP996426 KP996474 42 National Forest. J. Starr 10S-033 & T. Villaverde. 26/7/2010. (CAN)

174 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. arctogena COL 39.26528 ΫͳͲͷǤͷʹͲ͸͹ USA, Colorado, Lake Co., San Isabel arctogena B — KP996335 KP996424 KP996472 43 National Forest. J. Starr 10S-030 & T. Villaverde. 25/7/2010. (CAN) C. arctogena COL 39.26528 ΫͳͲͷǤͷʹͲ͸͹ USA, Colorado, Lake Co., San Isabel arctogena B — KP996336 KP996425 KP996473 44 National Forest. J. Starr 10S-030 & T. Villaverde. 25/7/2010. (CAN) C. arctogena MNT 45.05800 ΫͳͲͻǤͶ͹͸ͲͲ USA, Montana, Carbon Co. H. arctogena B — KP996334 KP996423 — 33 Lackshewitz 7790. 11/8/1977. (WTU- 288770) C. arctogena MNT 45.03203 ΫͳͲͺǤͷͳͶͻ͹ USA, Montana, Carbon Co., Custer arctogena B — KP996339 KP996428 KP996476 34 National Forest J. Starr 10S-047A & T. Villaverde. 31/7/2010. (CAN) C. arctogena MNT 44.97142 ΫͳͲͺǤͷ͹ͻʹͺ USA, Wyoming, Park Co. J. Starr 10S- arctogena B — KP996340 KP996429 — 35 047B & T. Villaverde. 31/7/2010. (CAN) C. arctogena NEV 40.81100 ΫͳͳͲǤ͵͵ͳͲͲ USA, Utah, Duchesne Co., Uinta arctogena B — KP996344 KP996433 KP996479 38 Mountains. E. Lewis 512. 15/8/1955. (CAN-515168) C. arctogena ORE 44.11467 ΫͳʹͲǤ͵͹ͷͻͶ USA, Oregon, Deschutes Co., arctogena B — KP996325 KP996414 KP996467 27 Deschustes National Forest. J. Starr 10S-057 & T. Villaverde. 9/8/2010. (CAN) C. arctogena ORE 44.11400 ΫͳʹͳǤ͸ʹʹͲͲ USA, Oregon, Deschutes Co. C. Halpern arctogena B — KP996321 KP996410 — 28 159046 & T. Magee. 30/8/1982. (OSC- 159046)

175 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. arctogena ORE 44.11467 ΫͳʹͲǤ͵͹ͷͻͶ USA, Oregon, Deschutes Co., arctogena B — KP996324 KP996413 KP996466 50 Deschustes National Forest. J. Starr 10S-057 & T. Villaverde. 9/8/2010. (CAN)

C. arctogena UTA 40.77800 ΫͳͳͲǤʹ͵͹ͲͲ USA, Utah, Duchesne Co., Gilbert arctogena B — KP996331 KP996420 KP996470 36 Creek. A. Huber 440 & S. Goodrich. 25/8/1993. (BRY-368578) C. arctogena NEV 39.38800 ΫͳͳͻǤ͹ͻʹͲͲ USA, Nevada, Washoe Co. M. E. Lewis arctogena B — KP996345 KP996434 — 37 448. 17/7/1955. (CAN-550536) C. arctogena NWH 42.27083 Ϋ͹ͳǤ͵ͲͷͷͲ USA, New Hampshire, Alpine Garden, arctogena B — — KP996373 — 22 Mt. Washington. W. W. Eggleston 1681. 29/7/1989. (RM-23379) C. arctogena WAS 48.84233 ΫͳʹͲǤͳͶʹʹͷ USA, Washington, Whatcom Co. J. Starr arctogena B — KP996342 KP996431 KP996478 31 10S-061 & T. Villaverde. 11/8/2010. (CAN) C. arctogena WAS 48.84233 ΫͳʹͲǤͳͶʹʹͷ USA, Washington, Whatcom Co. J. Starr arctogena B — KP996341 KP996430 KP996477 32 10S-061 & T. Villaverde. 11/8/2010. (CAN) C. arctogena WYO 45.05200 ΫͳͲͻǤͷ͹ͶͲͲ USA, Wyoming, Park Co., Beartooth arctogena B — KP996332 KP996421 KP996471 29 Plateau. B. Neely s.n. 18/8/1984. (COLO-399492) C. capitata MAG 66.00000 171.00000 Russia, Chukotski Peninsula. U. P. capitata A — KP996304 KP996393 — 1 Kozhevnikov et al. s.n. 27/7/1970. (DAO-139887) C. capitata MAG 68.00000 167.00000 Russia, Chukotka, Anui upland region. capitata A — KP996308 KP996397 — 2 E. V. Zimarskaja et al. s.n. 12/7/1967. (DAO-139880)

176 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. capitata MAG 68.00000 167.00000 Russia, Western Chukotka. E. V. capitata A — KP996310 KP996399 — 3 Zimarskaja et al. s.n. 12/7/1967. (BRY-122530)

C. capitata YAK 69.41667 130.66667 Russia, Sakha Republic, Bulnsk region. capitata A — KP996352 — — 4 B. Yurtsev s.n. 25/6/1960. (DAO- 257437) C. capitata ALB 58.98333 ΫͳͳͲǤͳ͸͸͸͹ Canada, Alberta, Ft. Fitzgerald. W. J. capitata B — KP996300 KP996389 — 19 Cody 4533 & C. C. Loan. 19/7/1950. (RM-228683) C. capitata ASK 68.06300 ΫͳͶͷǤͲͻ͵ͲͲ USA, Alaska, Old John Lake Area. K. capitata B — KP996312 KP996401 KP996456 23 Holmen 61-1227. 13/7/1961. (CAN- 271116) C. capitata BRC 52.46667 ΫͳʹͶǤ͸ͺ͵͵͵ Canada, British Columbia, Anahim capitata B — KP996301 KP996390 — 21 Lake. J. Calder 18578 et al. s.n. 9/7/1956. (COLO-158463) C. capitata BRC 52.46667 ΫͳʹͶǤ͸ͺ͵͵͵ Canada, British Columbia, Anahim capitata B — KP996307 KP996396 — 22 Lake. J. Calder 18578. J. A. Parmelee & R. L. Taylor s.n. 9/7/1956. (WTU- 197744) C. capitata FIN 7 69.08800 21.92800 Finland, Enontekiö lapland. H. Väre capitata B KP984470 — — — 11515. 29/7/2001. (H-737814) C. capitata FIN 8 66.36700 29.53300 Finland, Kuusamo, Liikasenvaara. T. capitata B — KP996316 KP996405 KP996460 Ulvinen s.n. 9/8/1962. (CAN-276804) C. capitata FIN 9 66.36700 29.53300 Finland,Kuusamo, Liikasenvaara. T. capitata B — KP996305 KP996394 — Ulvinen s.n. 9/8/1962. (CAN-276804) C. capitata ICE 65.14400 ΫͳͶǤ͵ͻͶͲͲ Iceland, Hallormsstadhur. H. F. capitata B KP984472 — — — 15 Gotzsche HFG81-37. 22/7/1981. (C- 15/2009)

177 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. capitata ICE 66.05000 Ϋʹ͵Ǥͳ͵͵ͲͲ Iceland, Lagarfoss, Hróarstungu, capitata B — KP996306 KP996395 — 16 Lagarfljótsrannsóknir s.n. 26/6/1976. (ICEL-04081)

C. capitata ICE 66.00000 ΫͳͺǤ͵ͺ͵ͲͲ Iceland, Hrísey, Eyjafirði. A. capitata B — KP996298 KP996386 — 17 Garðarsson s.n. 12/8/1967. (ICEL- 04078) C. capitata ICE 65.65700 Ϋͳ͸ǤͺͳͷͲͲ Iceland, Dalfjall, Mývatnssvei. E. capitata B — KP996299 KP996388 — 18 Einarsson s.n. 21/8/1974. (ICEL- 04073) C. capitata MAG 62.20000 33.78333 Russia, Magadan region, North Even capitata B — KP996314 KP996403 KP996458 5 area. A.P. Hohrjakov s.n. 2/8/1976. (CAN- 455497) C. capitata MAN 58.63500 -94.13000 Canada, Manitoba, Churchill, south of capitata B — — KP996387 — 23 Fort. K. Johnson J73-402. 26/8/1973. (WIN-33557) C. capitata MAN 58.74700 ΫͻͶǤͳ͸ͷͲͲ Canada, Manitoba, Fort Churchill. J. capitata B — KP996292 KP996380 — 24 Shay 83-60. 11/7/1983. (WIN-40808) C. capitata MAN 58.75500 ΫͻͶǤͲ͹ͺͲͲ Canada, Manitoba, Fort Churchill. J. capitata B — KP996294 KP996382 — 25 Ritchie 2104. 5/8/1956. (WIN-22433) C. capitata MAN 57.83000 ΫͻʹǤͺͲͶ͵Ͳ Canada, Manitoba, Wapusk National capitata B — KP996296 KP996384 — 26 Park. E. Punter 03-509 & M. Piercey- Normore. 19/7/2003. (WIN-71429) C. capitata MAN 58.63700 Ϋͻ͵Ǥͺʹ͹ͲͲ Canada, Manitoba, Twin Lakes. A. Ford capitata B — KP996297 KP996385 — 27 02379 et al. 25/7/2002. (WIN-71024) C. capitata MAN 58.75500 ΫͻͶǤͲ͹ͺͲͲ Canada, Manitoba, Fort Churchill. J. capitata B — KP996313 KP996402 KP996457 29 Ritchie 2104. 5/8/1956. (CAN- 248387)

178 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. capitata MAN 58.76667 Ϋͻ͵Ǥͺ͵333 Canada, Manitoba, vicinity of capitata B — KP996315 KP996404 KP996459 30 Churchill. W. Schofield 6862 & H. Crum. 21/7/1956. (CAN-247332)

C. capitata NOR 60.07900 10.03300 Norway, Folldal Gammelsetran. M. capitata B KP984473 — — — 14 Vileid s.n. 18/8/1998. (O-235091) C. capitata NWT 65.71700 ΫͳͳͺǤͺ͵͵ͲͲ Canada, Northwest Territories, capitata B — KP996291 KP996379 — 35 Sawmill Bay. H. T. Shacklette 2970. 13/7/1948. (CAN-199991) C. capitata NWT 67.33333 ΫͳʹͷǤͷͺ͵͵͵ Canada, Northwest Territories, Aubry capitata B — KP996318 KP996407 KP996461 36 Lake. R. Riewe 225 & M. G. Marsh. 17/7/1976. (CAN-433230) C. capitata ONT 55.13420 ΫͺʹǤ͵ͳʹͺͲ Canada, Ontario, Kenora District. J. L. capitata B — KP996302 KP996391 — 30 Riley 5848. 23/8/1976. (CAN- 409561) C. capitata ONT 55.98000 Ϋͺ͹Ǥ͸ͶͶͲͲ Canada, Ontario, Fort Severn. I. capitata B — KP996351 — — 31 Hustich 1296. 13/7/1956. (CAN- 242845) C. capitata RUN 62.20000 33.78333 Russia, Karelia Republic. Ruuhijävi 40- capitata B — KP996309 KP996398 — 6 02. 9/7/2002. (H-744530) C. capitata SAS 54.06667 ΫͳͲͶǤͲͷͻͶͶ Canada, Saskatchewan, Waskesim. J. capitata B — KP996311 KP996400 KP996455 37 Hudson 5063. 31/7/1992. (CAN- 565528) C. capitata SWE 68.32700 18.83800 Sweden, Torne, Gemeinde Kiruna. H. capitata B KP984477 — — — 10 Hertel 22918. 8/8/1980. (M- 0151931) C. capitata SWE 63.18100 14.75100 Sweden, Jämtland, Paroecia Frösö. E. capitata B KP984476 — — — 11 Asplund s.n. 2/6/1925. (C15-2009N2)

179 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. capitata SWE 68.56667 19.50000 Sweden, Soland, Torne. C. G. Alm s.n. capitata B KP984475 — — — 12 9/8/1958. (V-539346) C. capitata SWE 68.56667 18.34167 Sweden, Torne Lappmark, Abisko. G. capitata B — KP996303 KP996392 — 13 Alm s.n. 6/8/1958. (H-1226056)

C. capitata YUK 64.36700 Ϋͳ͵͹Ǥʹ͸͹ͲͲ Canada, Yukon Territory, Dempster capitata B — KP996293 KP996381 — 32 Highway. R. Porsild 1593. 17/7/1968. (CAN-318505) C. capitata YUK 64.36700 Ϋͳ͵͹Ǥʹ͸͹ͲͲ Canada, Yukon, Ogilvie Mountains. A. capitata B — KP996317 KP996406 — 33 E. Porsild 1462 & R. Porsild. 28/6/1968. (CAN-318349) C. capitata YUK 61.86667 Ϋͳ͵ͷǤͺͺ͵͵͵ Canada, Yukon, Dawson. J. Calder capitata B — KP996295 KP996383 — 34 25796 & J. Gillett. 22/6/1960. (ALA- 43436) C. mono- KEN ΫͲǤͳ͸ʹͷ͸ 37.20828 Kenya, Mt. Kenya National Park. Naro mono- — KP996274 — KP996440 stachya 1 Moru route. M. L. Buide 114UPO-K. stachya 28/7/2007. (UPOS3304-111) C. mono- KEN ΫͲǤͳ͸ͷ͸ͳ 37.24197 Kenya, Mt. Kenya National Park, Naro mono- — KP996273 — — stachya 2 Moru route. M. L. Buide 114UPO-K. stachya 28/7/2007. (UPOS3306-462) C. oreophila TUR 37.75100 44.31600 Turkey. Hakkari Province, Kara Dag. oreophila KP984462 — — KP996441 Davis & Polunim 24438. 16/8/1954. (E-00353688) C. obtusata WYO 44.98930 ΫͳͳͲǤ͹͸͸͹ USA, Wyoming, Park Co., Yellowstone Outgroup KP984459 KP996270 — KP996437 1 National Park. E. F. Evert 38901. 9/7/2001. (MOR0060897-164295)

180 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Coordinates GenBank accession numbers Pop. Species code Latitude Longitude Voucher Clade ITS atpF–atpH matK rps16

C. obtusata WYO 43.81930 ΫͳͲͻǤͲ͸͵͵ USA, Wyoming, Hot Springs Co., Outgroup KP984460 KP996271 — KP996438 2 Shoshone National. Forest. E. Evert 39259. 2/7/2002. (MOR-0060899- 162917) C. obtusata WYO 43.77250 ΫͳͲͻǤͳͻͺͷͲ USA, Wyoming, Hot Springs Co., Outgroup KP984461 KP996272 — KP996439 3 Absaroka Mountains. E. Evert 38141. 29/6/2000. (MOR-0060898-161081)

C. rupestris SPA 42.68350 0.07240 Spain, Huesca, Parque Nacional de Outgroup — KP996269 KP996356 — Ordesa y Monte Perdido. M. L. Buide 57MBR04 & J. M. Marín. 30/7/2004. (UPOS-168) C. runssoro- KEN ΫͲǤͳ͵͵͸ͻ 37.23439 Kenya, Mt. Kenya National Park, Naro runssoro- — KP996275 — — ensis Moru route. M. L. Buide et al. 113UPO- ensis K. 28/7/2007.(UPOS3305-461) Uncinia ARG Ϋͷ͵ǤͳͷͻͷͲ Ϋ͹ͳǤͳ͹͸ͳͲ Chile, Punta Arenas, Reserva forestal Outgroup — KP996268 KP996355 — triquetra de Magallanes. M. Luceño 185ML05 & R. Álvarez. 28/12/2005. (UPOS-1803)

181 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Table S2 Characteristics of the DNA regions sequenced for complete datasets including all species in Carex sections Capituligerae and Longespicatae and outgroups. Primers ITS-4 / ITS-A atpF / atpH matk 2.1f_J /matk 5r_J rps16F / rps16R

Description of regions Internal transcribed Intergenic spacer Chloroplast gene of a maturase protein Intergenic spacer spacers 1 and 2 and of chloroplast of chloroplast 5.8S ribosomal RNA region region References White (1990) and Fazekas et al. Plant Working Group, Royal Botanical Gardens Shaw et al. Blattner (1999) (2008) Kew, http://www.kew.org/barcoding/protoc (2005) ols.html, modified by Chouinard (2010) Total number of sequen- 19 87 89 49 ces in the alignment Aligned length (bp) 628 605 746 856 Ungapped length range 616–624 516–601 693–746 643–856 Identical sites 571 (91.8%) 579 (97.7%) 722 (96.8%) 833 (97.3%) Pairwise identity 64.50% 99.30% 99.50% 99.50% Variable characters 51 17 21 34 Parsimony-informative 36 14 12 14 characters Number of informative 13 1 0 0 indels Mean G+C content 47.90% 27.90% 28.90% 27.10% Substitution model HKY+I (ITS 1)/ JC HKY HKY+I GTR (5.8S)/ GTR (ITS 2)

182 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena

Table S3 Locus information for the regions amplified in the study including samples sizes, summary statistics. Nh, number of haplotypes (gaps were excluded); Hd, haplotype diversity; Sǡ•‡‰”‡‰ƒ–‹‰•‹–‡•ǢɎǡucleotide diversity.

Locus Nh Hd S Ɏ Tajima’s D Fu & Li’s D* Fu & Li’s F* atpF–atpH 5 0.725 8 0.00442 1.27151n.s. 1.27758n.s. 1.50645n.s. rps16 4 0.729 4 0.00245 1.63220n.s. 1.01718n.s. 1.40374n.s. matK 6 0.746 8 0.00397 1.85050n.s. 1.28088n.s. 1.73597*

*P < 0.05; n.s., not significant.

Table S4 Average within-group nucleotide substitution estimates for the matK gene of the complete dataset. dS, number of synonymous sites; dN, number of non-synonymous sites.

Selection hypothesis tested

Neutrality Positive Purifying

Species Probability †ΫdN Probability dSΫdN Probability dSΫdN

Outgroups 0.508n.s. ΫͲǤ͸͸Ͷ 1.000n.s. ΫͲǤ͸͸͹ 0.252n.s. 0.670 C. monostachya 0.040* 2.081 0.019* 2.106 1.000n.s. ΫʹǤͲͻ͵ and C. runssoro- ensis C. capitata A 1.000n.s. 0 .000 1.000n.s. 0.000 1.000n.s. 0.000 C. capitata B 0.304n.s. 1.031 0.154n.s. 1.022 1.000n.s. ΫͳǤͲ͵ͺ C. arctogena A 1.000n.s. 0.000 1.000n.s. 0.000 1.000n.s. 0.000 C. arctogena B 0.297n.s. ΫͳǤͲͶͺ 0.150n.s. 1.039 1.000n.s. ΫͳǤͲ͵Ͳ

*P < 0.05; n.s., not significant.

183 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Table S5 Loadings matrix obtained by the principal components analysis not corrected by phylogeny of 19 bioclimatic variables on Carex sections Capituligerae and Longespicatae.

Variable PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PC11 PC12 PC13 PC14 PC15 PC16 PC17 PC18 PC19 bio1 ΫͲǤʹͺ͸ ΫͲǤʹͳͻ ΫͲǤͲ͸ͻ 0.217 ΫͲǤͲͷʹ ΫͲǤͳͶͷ ΫͲǤͲͻͳ ΫͲǤͳͻͳ ΫͲǤͳͲͷ ΫͲǤͳͳͲ 0.286 ΫͲǤͷ͵ͳ 0.062 ΫͲǤͲʹͻ 0.377 ΫͲǤͲͳͻ 0.464 0.033 0.000 bio2 ΫͲǤͳͳͲ ΫͲǤʹͻͶ 0.187 ΫͲǤͲ͵͹ 0.570 0.339 0.330 0.076 ΫͲǤͲ͸ʹ ΫͲǤ͵͹ͺ 0.008 ΫͲǤͲͷͺ 0.045 0.145 ΫͲǤ͵ͳ͸ ΫͲǤͲͷ͵ 0.185 0.009 0.000 bio3 ΫͲǤ͵Ͳͺ ΫͲǤͲͻ͵ ΫͲǤͲʹ͹ 0.093 0.053 0.601 0.083 ΫͲǤͲ͸ͳ ΫͲǤͳ͵͸ 0.652 0.150 0.023 ΫͲǤͳʹͶ ΫͲǤͲͺͲ 0.105 0.000 ΫͲǤͳͳʹ ΫͲǤͲͳͻ 0.000 bio4 0.328 0.009 0.209 ΫͲǤͲͳͺ 0.113 ΫͲǤͳͳʹ ΫͲǤͲͷͻ 0.035 0.071 0.394 0.064 0.058 ΫͲ.007 0.021 ΫͲǤͳ͸ʹ ΫͲǤͲͷͳ 0.447 0.650 0.000 bio5 ΫͲǤͲ͸ͻ ΫͲǤ͵͹ͷ 0.209 0.157 0.273 ΫͲǤͳͻͶ ΫͲǤͳͲͳ ΫͲǤͲ͹ʹ ΫͲǤͲͲ͹ 0.008 ΫͲǤ͵ͷͶ 0.395 ΫͲǤͳͳ͵ ΫͲǤ͵ʹ͹ 0.379 0.100 0.021 ΫͲǤͲͳ͹ ΫͲǤ͵ʹ͵ bio6 ΫͲǤ͵ʹʹ ΫͲǤͳͲͷ ΫͲǤͳ͹͵ 0.083 ΫͲǤͳ͸͵ ΫͲǤͲͷͶ ΫͲǤͲͷ͵ 0.014 ΫͲǤͳͲ͸ ΫͲǤͲͶ͵ ΫͲǤʹͲ͹ 0.400 ΫͲǤͲͷ͹ ΫͲǤͲͺͶ ΫͲǤͳ͸Ͳ ΫͲǤͲʹͻ 0.331 ΫͲǤͲͳͳ 0.674 bio7 0.293 ΫͲǤͲ͹ͷ 0.277 ΫͲǤͲͲͺ 0.297 ΫͲǤͲ͵ͻ 0.004 ΫͲǤͲͶͻ 0.104 0.048 0.038 ΫͲǤʹͳͶ 0.003 ΫͲǤͲ͹͵ 0.347 0.078 ΫͲǤ͵ʹ͸ 0.003 0.665 bio8 0.152 0.008 0.125 0.658 ΫͲǤʹͺ͸ ΫͲǤͲ͵͵ 0.580 0.317 0.029 0.007 ΫͲǤͲ͸ͷ ΫͲ.020 0.018 ΫͲǤͲʹͷ 0.044 ΫͲǤͲͲͳ ΫͲǤͲͲͶ ΫͲǤͲͲ͵ 0.000 bio9 ΫͲǤʹͻͺ ΫͲǤͳͻͲ ΫͲǤͲͻ͹ ΫͲǤͳʹͳ 0.031 ΫͲǤͲ͸͵ ΫͲǤͲ͸ʹ 0.499 0.754 0.103 0.048 ΫͲǤͲͻͳ 0.028 ΫͲǤͲͶͷ 0.007 ΫͲǤͲͲͳ ΫͲǤͲͲͷ ΫͲǤͲͲʹ 0.000 bio10 ΫͲǤͲ͸Ͷ ΫͲǤ͵͸Ͷ 0.150 0.308 0.072 ΫͲǤͶͲͳ ΫͲǤʹʹͻ ΫͲǤͳͲ͵ ΫͲǤͲ͹Ͳ 0.274 0.180 ΫͲǤͲʹ͹ 0.033 0.282 ΫͲǤͶͷʹ ΫͲǤͲͺͳ ΫͲǤʹ͵͸ ΫͲǤʹ͵ͻ 0.000 bio11 ΫͲǤ͵ʹ͵ ΫͲǤͳ͵ʹ ΫͲǤͳ͵ͻ 0.107 ΫͲǤͲͺͲ ΫͲǤͲʹͷ ΫͲǤͲͳͷ ΫͲǤͲͶ͹ ΫͲǤͲͻͻ ΫͲǤʹʹʹ ΫͲǤͲ͵͹ ΫͲǤͲʹ͸ 0.045 0.080 ΫͲǤͲͲ͸ 0.072 ΫͲǤͶͻ͹ 0.718 0.000 bio12 ΫͲǤʹͶͶ 0.252 0.265 ΫͲǤͲͲͻ 0.028 ΫͲǤͳͲͶ 0.069 ΫͲǤͳͳʹ ΫͲǤͲͲ2 0.058 0.017 ΫͲǤͳͷʹ 0.359 ΫͲǤͷ͸ͺ ΫͲǤ͵͵͹ 0.430 ΫͲǤͲͳ͵ ΫͲǤͲͳͺ 0.000 bio13 ΫͲǤʹ͵ͳ 0.178 0.378 0.005 ΫͲǤͳͳ͹ 0.064 ΫͲǤͲʹͺ ΫͲǤͳ͸͸ 0.164 0.062 ΫͲǤͶͳͶ ΫͲǤͳͲͶ ΫͲǤͲͻͲ 0.575 0.083 0.391 0.115 ΫͲǤͲʹʹ 0.000 bio14 ΫͲǤͳͷͲ 0.351 ΫͲǤͳͲͳ 0.101 0.340 ΫͲǤʹͳͲ ΫͲǤͳͳͷ 0.405 Ϋ0.290 0.053 ΫͲǤͳ͵͸ ΫͲǤʹ͸ʹ ΫͲǤͷͷͺ ΫͲǤͲ͹ʹ ΫͲǤͲ͹ͳ 0.044 ΫͲǤͲͳ͵ 0.006 0.000 bio15 0.014 ΫͲǤͳ͸ͳ 0.496 ΫͲǤͲͻͷ ΫͲǤ͵͵Ͷ 0.230 ΫͲǤͶͲ͵ 0.501 ΫͲǤ͵Ͳͻ ΫͲǤͳͷͻ 0.112 0.001 0.063 ΫͲǤͲͶ͸ 0.035 ΫͲǤͲͲ͵ ΫͲǤͲͲͳ ΫͲǤͲͲ͵ 0.000 bio16 ΫͲǤʹ͵ͳ 0.172 0.384 ΫͲǤͲͶ͸ ΫͲǤͳͲ͹ ΫͲǤͲʹͷ 0.056 ΫͲǤʹͳͻ 0.137 ΫͲǤͲʹͳ ΫͲǤʹͳͳ ΫͲǤͳͶ͵ ΫͲǤͳͳ͹ ΫͲǤͳ͹͸ ΫͲǤͲͷ͸ ΫͲǤ͹ͷʹ ΫͲǤͲ͹ͳ 0.032 0.000 bio17 ΫͲǤͳͺʹ 0.344 ΫͲǤͲ͵Ͷ 0.120 0.318 ΫͲǤͲͻ͵ ΫͲǤͲͺʹ 0.231 ΫͲǤͳͷ͸ 0.118 ΫͲǤͲͶͲ 0.174 0.646 0.236 0.253 ΫͲǤʹ͵ͷ 0.006 ΫͲǤͲͳ͸ 0.000 bio18 ΫͲǤͲͶ͸ 0.346 0.141 0.420 0.127 0.179 ΫͲ.285 ΫͲǤͳͷ͸ 0.298 ΫͲǤʹ͹͵ 0.463 0.323 ΫͲǤʹͲ͹ ΫͲǤͲͲͶ 0.008 0.042 0.018 0.025 0.000 bio19 ΫͲǤʹͷͶ 0.048 0.244 ΫͲǤ͵ͺͳ ΫͲǤͲͷͲ ΫͲǤ͵͸Ͳ 0.441 0.044 ΫͲǤͳʹ͹ 0.039 0.467 0.286 ΫͲǤͳͺͳ 0.124 0.176 0.054 ΫͲǤͲʹʹ 0.000 0.000 % of 41.900% 26.940% 14.340% 7.699% 4.640% 2.338% 0.915% 0.511% 0.312% 0.213% 0.063% 0.053% 0.031% 0.017% 0.015% 0.010% 0.003% 0.001% 0.000% variance

184 ______Chapter 3. Direct long-distance dispersal best explains______the bipolar distribution of Carex arctogena

Table S6 Loadings matrix obtained by the principal components analysis corrected by phylogeny of 19 bioclimatic variables on Carex sections Capituligerae and Longespicatae.

Variables PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PC11 PC12 PC13 PC14 PC15 PC16 PC17 PC18 PC19 bio1 Ϋ0.286 Ϋ0.219 Ϋ0.069 0.217 Ϋ0.052 Ϋ0.145 Ϋ0.091 Ϋ0.191 Ϋ0.105 Ϋ0.110 0.286 Ϋ0.531 0.062 Ϋ0.029 0.377 Ϋ0.019 0.464 0.033 0.000 bio2 Ϋ0.110 Ϋ0.294 0.187 Ϋ0.037 0.570 0.339 0.330 0.076 Ϋ0.062 Ϋ0.378 0.008 Ϋ0.058 0.045 0.145 Ϋ0.316 Ϋ0.053 0.185 0.009 0.000 bio3 Ϋ0.308 Ϋ0.093 Ϋ0.027 0.093 0.053 0.601 0.083 Ϋ0.061 Ϋ0.136 0.652 0.150 0.023 Ϋ0.124 Ϋ0.080 0.105 0.000 Ϋ0.112 Ϋ0.019 0.000 bio4 0.328 0.009 0.209 Ϋ0.018 0.113 Ϋ0.112 Ϋ0.059 0.035 0.071 0.394 0.064 0.058 Ϋ0.007 0.021 Ϋ0.162 Ϋ0.051 0.447 0.650 0.000 bio5 Ϋ0.069 Ϋ0.375 0.209 0.157 0.273 Ϋ0.194 Ϋ0.101 Ϋ0.072 Ϋ0.007 0.008 Ϋ0.354 0.395 Ϋ0.113 Ϋ0.327 0.379 0.100 0.021 Ϋ0.017 Ϋ0.323 bio6 Ϋ0.322 Ϋ0.105 Ϋ0.173 0.083 Ϋ0.163 Ϋ0.054 Ϋ0.053 0.014 Ϋ0.106 Ϋ0.043 Ϋ0.207 0.400 Ϋ0.057 Ϋ0.084 Ϋ0.160 Ϋ0.029 0.331 Ϋ0.011 0.674 bio7 0.293 Ϋ0.075 0.277 Ϋ0.008 0.297 Ϋ0.039 0.004 Ϋ0.049 0.104 0.048 0.038 Ϋ0.214 0.003 Ϋ0.073 0.347 0.078 Ϋ0.326 0.003 0.665 bio8 0.152 0.008 0.125 0.658 Ϋ0.286 Ϋ0.033 0.580 0.317 0.029 0.007 Ϋ0.065 Ϋ0.020 0.018 Ϋ0.025 0.044 Ϋ0.001 Ϋ0.004 Ϋ0.003 0.000 bio9 Ϋ0.298 Ϋ0.190 Ϋ0.097 Ϋ0.121 0.031 Ϋ0.063 Ϋ0.062 0.499 0.754 0.103 0.048 Ϋ0.091 0.028 Ϋ0.045 0.007 Ϋ0.001 Ϋ0.005 Ϋ0.002 0.000 bio10 Ϋ0.064 Ϋ0.364 0.150 0.308 0.072 Ϋ0.401 Ϋ0.229 Ϋ0.103 Ϋ0.070 0.274 0.180 Ϋ0.027 0.033 0.282 Ϋ0.452 Ϋ0.081 Ϋ0.236 Ϋ0.239 0.000 bio11 Ϋ0.323 Ϋ0.132 Ϋ0.139 0.107 Ϋ0.080 Ϋ0.025 Ϋ0.015 Ϋ0.047 Ϋ0.099 Ϋ0.222 Ϋ0.037 Ϋ0.026 0.045 0.080 Ϋ0.006 0.072 Ϋ0.497 0.718 0.000 bio12 Ϋ0.244 0.252 0.265 Ϋ0.009 0.028 Ϋ0.104 0.069 Ϋ0.112 Ϋ0.002 0.058 0.017 Ϋ0.152 0.359 Ϋ0.568 Ϋ0.337 0.430 Ϋ0.013 Ϋ0.018 0.000 bio13 Ϋ0.231 0.178 0.378 0.005 Ϋ0.117 0.064 Ϋ0.028 Ϋ0.166 0.164 0.062 Ϋ0.414 Ϋ0.104 Ϋ0.090 0.575 0.083 0.391 0.115 Ϋ0.022 0.000 bio14 Ϋ0.150 0.351 Ϋ0.101 0.101 0.340 Ϋ0.210 Ϋ0.115 0.405 Ϋ0.290 0.053 Ϋ0.136 Ϋ0.262 Ϋ0.558 Ϋ0.072 Ϋ0.071 0.044 Ϋ0.013 0.006 0.000 bio15 0.014 Ϋ0.161 0.496 Ϋ0.095 Ϋ0.334 0.230 Ϋ0.403 0.501 Ϋ0.309 Ϋ0.159 0.112 0.001 0.063 Ϋ0.046 0.035 Ϋ0.003 Ϋ0.001 Ϋ0.003 0.000 bio16 Ϋ0.231 0.172 0.384 Ϋ0.046 Ϋ0.107 Ϋ0.025 0.056 Ϋ0.219 0.137 Ϋ0.021 Ϋ0.211 Ϋ0.143 Ϋ0.117 Ϋ0.176 Ϋ0.056 Ϋ0.752 Ϋ0.071 0.032 0.000 bio17 Ϋ0.182 0.344 Ϋ0.034 0.120 0.318 Ϋ0.093 Ϋ0.082 0.231 Ϋ0.156 0.118 Ϋ0.040 0.174 0.646 0.236 0.253 Ϋ0.235 0.006 Ϋ0.016 0.000 bio18 Ϋ0.046 0.346 0.141 0.420 0.127 0.179 Ϋ0.285 Ϋ0.156 0.298 Ϋ0.273 0.463 0.323 Ϋ0.207 Ϋ0.004 0.008 0.042 0.018 0.025 0.000 bio19 Ϋ0.254 0.048 0.244 Ϋ0.381 Ϋ0.050 Ϋ0.360 0.441 0.044 Ϋ0.127 0.039 0.467 0.286 Ϋ0.181 0.124 0.176 0.054 Ϋ0.022 0.000 0.000 % of 98.993% 0.786% 0.126% 0.052% 0.023% 0.008% 0.006% 0.004% 0.001% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% variance

185 ______Chapter 3. Direct lon______g-distance dispersal best explains the bipolar distribution of Carex arctogena

Table S7 Bioclimatic variables used. Units of bioclimatic variables are °C × 10 for temperature (excluding BIO4, which was calculated based on K × 10 to deal with negative temperatures) and mm for precipitation.

Bioclimatic variable Description

BIO1 annual mean temperature BIO2 mean diurnal temperature range [mean of monthly (maximum temperature Ϋ minimum temperature)] BIO3 isothermality (BIO2 / BIO7 × 100) BIO4 temperature seasonality (standard deviation of monthly temperature) BIO5 maximum temperature of the coldest month BIO6 minimum temperature of the warmest month BIO7 temperature range (BIO6 Ϋ BIO5) BIO8 mean temperature of the wettest quarter BIO9 mean temperature of the driest quarter BIO10 mean temperature of the warmest quarter BIO11 mean temperature of the coldest quarter BIO12 annual precipitation BIO13 precipitation of the wettest month BIO14 precipitation of the driest month BIO15 precipitation seasonality (coefficient of variation of monthly precipitation) BIO16 precipitation of the wettest quarter BIO17 precipitation of the driest quarter BIO18 precipitation of the warmest quarter BIO19 precipitation of the coldest quarter

REFERENCES

Blattner, F.R. (1999) Direct amplification of the (2008) Multiple multilocus DNA barcodes from entire ITS region from poorly preserved plant the plastid genome discriminate plant species material using recombinant PCR. Biotechniques, equally well. PLoS ONE, 3, 1–12. 27, 1180–1186. Shaw, J., Lickey, E.B., Beck, J.T., Farmer, S.B., Liu, W., Brummitt, R.K. (2001) World geographical scheme Miller, J., Siripun, K.C., Winder, C.T., Schilling, E., for recording plant distributions, 2nd ed. Hunt & Small, R.L. (2005) The tortoise and the hare Institute for Botanical Documentation, Pitts- II: relative utility of 21 noncoding chloroplast burgh, PA. DNA sequences for phylogenetic analysis. Am- Chouinard, B.N. (2010) DNA Barcodes for the Caric- erican Journal of Botany, 92, 142–166. eae (Carex & Kobresia, Cyperaceae) of North White, T.J., Bruns, T., Lee, S. & Taylor, J. (1990) America, north of Mexico. University of Ottawa, Amplification and direct sequencing of fungal Ottawa, ON. ribosomal RNA genes for phylogenetics. PCR Fazekas, A.J., Burgess, K.S., Kesanakurti, P.R., protocols: a guide to methods and applications Graham, S.W., Newmaster, S.G., Husband, B.C., (ed. by M. Innis, D. Gelfand, D. Sninsky and T. Percy, D.M., Hajibabaei, M. & Barrett, S.C.H. White), Academic Press.

186 Chapter 4

Long-distance dispersal during the middle–late Pleistocene explains the bipolar disjunction of Carex maritima (Cyperaceae)

187 188 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction of Carex maritima Journal of Biogeography (J. Biogeogr.) (2015)

ORIGINAL Long-distance dispersal during the ARTICLE middle–late Pleistocene explains the bipolar disjunction of Carex maritima (Cyperaceae) Tamara Villaverde1*, Marcial Escudero2, Modesto Luceno~ 1 and Santiago Martın-Bravo1

1Botany area, Department of Molecular ABSTRACT Biology and Biochemical Engineering, Pablo Aim We set out to explain the bipolar distribution of Carex maritima, clarify- de Olavide University, Seville, Spain, 2 ing the direction and timing of dispersal. We also tested mountain-hopping Department of Integrative Ecology, Estacion Biologica de Donana~ (EBD – CSIC), Seville, and direct long-distance dispersal hypotheses, as well as the relationship of C. Spain maritima with biotic and abiotic factors that could explain the bipolar distribu- tion. Location Arctic/boreal latitudes of both hemispheres. Methods Molecular and bioclimatic data were obtained for C. maritima and related species from section Foetidae. We sequenced two (rps16 and 50trnK intron) plastid DNA regions (cpDNA) and the external and internal tran- scribed spacers (ETS and ITS) of the nuclear ribosomal gene region (nrDNA) and inferred phylogenetic relationships, divergence time estimates and biogeo- graphical patterns using maximum likelihood, statistical parsimony, Bayesian inference and ecological niche modelling.

Results Carex maritima populations from the Southern Hemisphere were genetically and ecologically differentiated from their northern counterparts and formed a monophyletic group nested within a paraphyletic C. maritima. Diver- gence time analysis estimated a middle–late Pleistocene divergence of the southern lineage (0.23 Ma; 95% highest posterior density: 0.03–0.51 Ma). Southern Hemisphere populations are more stenotopic than the Northern Hemisphere ones, which tolerate harsher conditions. Main conclusions Our results point to a middle–late Pleistocene migration of C. maritima by long-distance dispersal, either directly or via mountain-hop- *Correspondence: Tamara Villaverde, Botany area, Department of Molecular Biology and ping, from the Northern Hemisphere to the Southern Hemisphere. Biochemical Engineering, Pablo de Olavide Keywords University, ctra. de Utrera km 1 s/n, 41013 Seville, Spain. Biogeography, bipolar distribution, Carex, climatic niche, Cyperaceae, E-mail: [email protected] divergence time estimation, Foetidae, long-distance dispersal.

mountain ranges spread throughout the world. He invoked INTRODUCTION signs of an Ice Age in the high latitudes of the Northern Darwin (1872) studied some potential mechanisms underly- Hemisphere to argue that these plants could have migrated ing the disjunctions of arctic–alpine plant species to refute southwards and descended from the mountain summits dur- the idea of multiple creations (Gmelin, 1747) in favour of ing the glacial epoch. As the climate subsequently warmed the hypothesis of a single origin and subsequent migrations. up in the high latitudes of the Northern Hemisphere, plants He compiled evidence about the time in which these plants would have recolonized northwards, as well as moved to could have initiated their migrations, together with the higher elevations in the mountainous regions of the lower means and directions of colonization (e.g. seed survival in latitudes of the Northern Hemisphere. When the Southern oceans or seed dispersal by birds), making use of data to Hemisphere experienced a glacial period, these isolated pop- explain the similarities between the floras of very distant ulations would have been able to spread, in time reaching

ª 2015 John Wiley & Sons Ltd http://wileyonlinelibrary.com/journal/jbi 1 doi:10.1111/jbi.12559 189 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction of Carex maritima T. Villaverde et al. the scattered locations in the high latitudes of the Southern tiation between Northern and Southern Hemisphere Hemisphere where they persist. Nearly a century and a half populations of C. maritima, no North American populations later, the ideas and hypotheses proposed by Darwin to were included in their analyses. explain bipolar disjunctions are still admired as having been Although the vicariance hypothesis (Du Rietz, 1940) has well ahead of their time (Donoghue, 2011). Nowadays, a traditionally been considered in explanation of Carex bipolar combination of bioclimatic and molecular data can help us distribution (e.g. Villaverde et al., 2015), it can now easily be to better assess the possible evolutionary, climatic and geo- rejected for Carex bipolar species as the age of the diversifi- logical changes at the origin of the biogeographical patterns cation of the Cyperaceae family is younger than the trans- (Crisp et al., 2011). tropical highland bridges (82.6 Ma, 95% highest posterior Historical biogeography has been dominated over the past density, HPD: 75.9–85.6 Ma; Escudero & Hipp, 2013). We few decades by investigations of shared distribution patterns can also discard parallel evolution for C. maritima because among taxa (e.g. Sanmartın et al., 2001; Posadas et al., Escudero et al. (2010a) showed that populations from the 2006). Organism distributions in the Southern Hemisphere, Northern Hemisphere and the Southern Hemisphere were together with the underlying causes, have long been analysed part of the same clade. (e.g. Raven, 1963; Raven & Axelrod, 1974; Wen & Ickert- The aim of the present study was to explain the bipolar Bond, 2009; reviewed in Sanmartın & Ronquist, 2004). distribution of C. maritima. Specifically, our aims were: (1) One of the most fascinating plant distribution patterns to clarify the direction of the dispersal (north-to-south or concerning the Southern Hemisphere encompasses the bipo- south-to-north); (2) in the case of genetic structure, to lar disjunction (> 55 N and > 52 S), achieved only by c.30 estimate the timing of dispersal; and (3) to test mountain- vascular plant species (Moore & Chater, 1971). Four hypoth- hopping and direct long-distance dispersal hypotheses, as eses have historically been put forward to account for bipolar well as the relationship of C. maritima with biotic and abi- disjunctions: (1) convergent or parallel evolution of the dis- otic factors that could explain the bipolar distribution. In junct populations (Scotland, 2011); (2) vicariance (Du Rietz, order to accomplish this task we combined a wide sampling 1940), which implies a continuous distribution fragmenta- of the species’ range with data from nuclear and plastid tion dating back to the trans-tropical highland bridges dur- molecular markers and bioclimatic data. We analysed the ing the Mesozoic Era (from the early Jurassic, 195 Ma; phylogenetic and phylogeographical relationships of C. mari- Scotese et al., 1988); (3) stepwise long-distance dispersal tima populations and compared its ecological niche through- across the equator and via mountain ranges (‘mountain-hop- out its distribution. ping’; Raven, 1963; Moore & Chater, 1971; Ball, 1990; Heide, 2002; Vollan et al., 2006); and lastly (4) direct long-distance MATERIALS AND METHODS seed dispersal by birds, wind and/or ocean currents (Cruden, 1966; Munoz~ et al., 2004; Nathan et al., 2008). Sampling Six out of the c. 30 bipolar vascular plant species known belong to the genus Carex L. (Moore & Chater, 1971), a spe- We obtained plant material representing the geographical cies-rich genus (> 2000 species) found especially in the tem- range of C. maritima (42 populations) as circumscribed by perate and cold regions of the Northern Hemisphere Egorova (1999). Samples used for the molecular study were (Reznicek, 1990). Molecular studies focused on bipolar Carex obtained from fresh leaf material collected in the field and species (Vollan et al., 2006; Escudero et al., 2010a; Villaverde dried in silica gel, and from herbarium specimens (see et al., 2015) determined low levels of genetic differentiation Appendix S1 in Supporting Information). Vouchers for new between the disjunct populations, suggesting either moun- collections are deposited in CAN, COLO, SI and UPOS tain-hopping or direct long-distance dispersal, yet none of herbaria (abbreviations according to Index Herbariorum; these studies could determine which hypothesis best http://sciweb.nybg.org/science2/IndexHerbariorum.asp). We explained the observed distributions of the bipolar species. emphasized the sampling of the most northern Southern Carex maritima Gunn. [sect. Foetidae (Tuckerm. ex L.H. Bai- Hemisphere populations and we were able to obtain material ley) Kuk.]€ is an arctic–alpine species with a circumboreal dis- from northern parts of Argentina. We were not, however, tribution including the European Alps and the Himalayas in able to sample other more northerly populations in the the Northern Hemisphere, while in the Southern Hemisphere Southern Hemisphere, from Ecuador and Bolivia (Govaerts it is distributed from Ecuador to Patagonia (Govaerts et al., et al., 2014). We also included four other species from 2014; see Fig. 1). It is a wind-pollinated herbaceous hemi- Carex sect. Foetidae [10–15 species in total, including C. cryptophyte or rhizome geophyte, which generally colonizes maritima; Reznicek, 2002; eMonocot Cyperaceae (http:// water-influenced habitats (e.g. lake, river, ocean shores or cyperaceae.e-monocot.org, accessed 3 December 2014)]: C. snowmelt water areas) and hydromorphic soils (e.g. beaches, incurviformis Mack. (two populations), C. pseudofoetida Kuk.€ fens, alluviums). Recent morphological and taxonomical (two populations), C. sajanensis V. I. Krecz. (four popula- studies of C. maritima (Moore & Chater, 1971; Reznicek, tions) and C. vernacula L. H. Bailey (three populations). As 2002) did not reveal any infraspecific taxa. Although Escu- outgroups, we included taxa from the subgenus Vignea dero et al. (2010a) detected some degree of genetic differen- (P. Beauv. ex Lestib. f.) Perterm. (Hendrichs et al., 2004;

2 Journal of Biogeography ª 2015 John Wiley & Sons Ltd 190 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction of Carex maritima Bipolar disjunction in Carex maritima

Figure 1 Distribution map of the sampled populations of Carex maritima, C. incuviformis and C. pseudofoetida. Carex maritima populations (46; some populations overlap in the map, see Appendix S1 for more details) are depicted by black circles, white diamonds represent C. incurviformis (two populations) and grey triangles indicate C. pseudofoetida samples (two populations). The dashed region denotes the distribution of C. maritima, obtained from the World Checklist of Cyperaceae (Govaerts et al., 2014).

Escudero & Hipp, 2013): C. stenophylla Wahlenb. from sec- There are two main strategies for inferring phylogenies tion Divisae H. Christ ex Kuk.€ (three populations), C. remota from multiple DNA regions: (1) the total evidence approach, L. from section Remotae (Ascherson) C. B. Clarke (one pop- in which phylogeny is reconstructed from as much data as ulation), C. canescens L. from section Glareosae G. Don (one possible to obtain the dominant signal (Kluge, 1989); (2) population) and C. paniculata L. from section Heleoglochin gene-by-gene strategy, in which it is often possible to identify Dumort. (one population; see Fig. 1 and Appendix S1). For and explain gene tree incongruences (Rannala & Yang, all species, one individual per population was sampled except 2008). In the last few years, coalescent species tree methods for four populations of C. maritima, for which two individu- have been used to reconcile population history with incon- als were included (Yukon, Nunavut, Iceland and Argentina; gruent phylogenies derived from different DNA regions (De- see Appendix S1). gnan & Rosenberg, 2009). Because of the absence of incongruences between DNA regions (results not shown) and the little genetic variation found in them, we have used the PCR amplification and sequencing total evidence strategy in the current study. Total DNA was extracted using DNeasy Plant Mini Kit (Qia- Maximum likelihood analyses of the unpartitioned com- gen, Valencia, CA, USA). Forward and reverse primers were bined matrix were performed using RAxML 7.2.6 (Stamata- used for amplifications of the internal transcribed spacer kis, 2006) with a GTR-GAMMA model of sequence (ITS) region (ITS-A, ITS-4; White et al., 1990; Blattner, evolution and node support assessed with 1000 bootstrap 1999), external transcribed spacer (ETS) region (ETS-1f, 18S- (BS) replicates. Bayesian inference analyses were executed in R; Starr et al., 2003), 50trnK intron (50trnKCarexF, 50trnKCa- MrBayes 3.2 (Ronquist et al., 2012). The most appropriate rexR; Escudero & Luceno,~ 2009) and rps16 intron (rps16– nucleotide substitution model for each partition was chosen rps16R; Shaw et al., 2005). Thermal cycling was carried out using the Akaike information criterion (AIC) in jModelTest in a Perkin Elmer PCR-system 9700 (Foster City, CA) under (Posada, 2008). Selected nucleotide substitution models were the conditions specified by Escudero et al. (2010a) for ITS GTR+I, HKY and GTR+G for ITS1, 5.8S and ITS2, respec- and rps16; and Starr et al. (2003) and Escudero & Luceno~ tively; HKY+I for ETS; F81 + I for 50trnK and GTR for rps16 (2009) for the ETS region and 50trnK intron, respectively. (Appendix S1). The Markov chain Monte Carlo (MCMC) Polymerase chain reaction (PCR) products were cleaned and search was run for five million generations with a tree sam- sequenced following Escudero et al. (2008). Sequences were pled every 1000 generations and two simultaneous analyses edited, automatically aligned with muscle (Edgar, 2004) and started from different random trees (Nruns = 2), each with manually adjusted using Geneious 6.1.7 (Biomatters, Auck- four Markov chains (Nchains = 4). The first 20% of the trees land, New Zealand). were discarded from each run as the burn-in. A Bayesian majority-rule consensus tree was calculated in MrBayes with posterior probability (PP) values as a measure for clade sup- Phylogenetic and haplotype analyses port. We used a total of 44 sequences of ITS (six from GenBank), We estimated the genealogical relationships among the 43 of ETS (one from GenBank), 48 of 50trnK (one from two cpDNA haplotypes using the plastid 50trnK–rps16 matrix GenBank) and 51 of rps16 (two from GenBank; see Appen- and statistical parsimony as implemented in tcs 1.21 (Clem- dix S1). Each locus was analysed independently and in com- ent et al., 2000). Owing to the polyphyly of the section (see bination using maximum likelihood (ML) and Bayesian Results), this analysis was only performed for the core Foeti- inference (BI). The combined nuclear and plastid aligned dae, which comprises all sampled members of section Foeti- matrix consisted of 197 sequences from 64 individuals and dae except for C. vernacula. The maximum number of 2699 sites (Appendix S1). differences resulting from single substitutions among

Journal of Biogeography 3 ª 2015 John Wiley & Sons Ltd 191 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction of Carex maritima T. Villaverde et al. haplotypes was calculated with 95% confidence limits. Two lineages. We represented data associated with the most informative indels in 50trnK and one in rps16 were coded as important bioclimatic variables retained in the phylogenetic a presence–absence character for analysis. Gaps due to PCA for C. maritima in boxplots. In order to compare cli- mononucleotide repeat units (poly-T and poly-A), which are mate regime similarities and differences of the species of core considered to be highly homoplasic (Kelchner, 2000), were Foetidae we included the samples of C. incurviformis, C. treated as missing data. We estimated completeness of haplo- pseudofoetida and C. sajanensis in the PCA of the climatic type (50trnK–rps16) sampling using a Stirling probability dis- environment. tribution as described by Dixon (2006), which calculates a posterior probability distribution of the total number of Past and present distribution under climatic change haplotypes (sampled or not). scenarios

Species distribution modelling was performed to reconstruct Divergence time estimation the potential ranges of C. maritima under present climatic Dated phylogenies were estimated for the combined nuclear conditions and for two historical periods, the Last Intergla- and plastid matrix in beast 1.7.5 (Drummond et al., 2012). cial (LIG; 120–140 ka) and the Last Glacial Maximum Phylogeny was estimated using an uncorrelated lognormal (LGM; 21 ka), with Maxent 3.3.3k (Phillips et al., 2006). relaxed clock model. A normal age prior with a mean of Neither C. incurviformis nor C. pseudofoetida was included in 14.82 Ma 2.5 Myr was applied to the root of the tree the model with C. maritima because their different ecological based on the previous estimate for the divergence of the sub- requirements may confound C. maritima distribution model- genus Vignea (Escudero & Hipp, 2013). Analyses were con- ling. Carex incurviformis is distributed only in North America ducted using two independent MCMC runs of 60 million and its ecology differs from that of C. maritima in this generations each, assuming the birth–death tree prior with a region (Reznicek, 2002). Moreover, the partial molecular dif- mean substitution rate set at 1.0. Run convergence and ferentiation (see Results) and their distinctive morphology burn-in were assessed in Tracer 1.5 (Rambaut & Drum- (Reznicek, 2002) also support this decision. Settings were mond, 2009). Maximum clade credibility trees were calcu- established following Blanco-Pastor et al. (2013). We per- lated with TreeAnnotator 1.7.2 (Drummond & Rambaut, formed a correlation analysis with the variance inflation fac- 2007) using a posterior probability limit of 0.7 and the mean tor (VIF) using the ‘vif’ function in the usdm package in R heights option. (R Core Team, 2014) and a correlation threshold of 0.7. Only three variables were uncorrelated and consequently included in the analyses: BIO1 (annual mean temperature), Climatic environment – ecological niche BIO6 (minimum temperature of the warmest month) and We obtained bioclimatic data for the localities of our molec- BIO12 (annual precipitation). Replicate runs (500) were per- ular sampling (56 samples of core Foetidae:47ofC. mariti- formed by using the bootstrap run type. All 19 of these vari- ma, two each of C. incurviformis and C. pseudofoetida, and ables have a grid size of 30 arc seconds for present and LGM five of C. sajanensis; ‘reduced data set’ from here on) in conditions but 2.5 arc minutes for LIG scenarios. These grid order to study the ecological factors influencing species’ size differences required us to omit 82 data points from the range. We compiled a new data set (‘full data set’) and also C. maritima full data set from the model, leaving a total of obtained bioclimatic data. This new data set was completed 812 points. Analyses were performed for all populations of by adding: (1) three additional populations from Austria, C. maritima and separately for Northern and Southern Italy and Sweden for which we failed to amplify any loci Hemisphere populations of C. maritima. We partitioned all (Appendix S1); and (2) species occurrence data between the locality data into training and testing data sets (75% vs. 1950 and 2000 downloaded from the Global Biodiversity 25%, respectively) in order to build niche models and to Information Facility data portal (http://www.gbif.org/, down- evaluate the quality of the model. Nonetheless, projections to loaded 22 December 2014) after pruning for likely incorrect past scenarios have to be interpreted with caution due to the identification or georeferencing (e.g. occurrences in oceans) absence of fossils to validate the model and the low number and removing duplicate records from the same locality to of existing localities in the Southern Hemisphere. Similar reduce the effects of spatial autocorrelation (847 new pres- results were obtained when modelling the climatic niche of ence data from preserved specimens of C. maritima). Finally, C. maritima using the reduced data set (results not shown). our full second data set included 894 populations in total (see Appendices S1 and S2). For each sampled population in RESULTS our data sets we obtained values for 19 bioclimatic variables (Appendix S1) as described by Escudero et al. (2013). We Phylogenetic reconstruction ran principal components analyses (PCA) using the full and reduced climatic data sets, as Villaverde et al. (2015). The BI and ML analyses revealed a lack of monophyly both for phylogenetic size-correction was performed in our reduced Carex sect. Foetidae and C. maritima. Strong support (100% data set for non-independence among the observations for BS/1 PP; Fig. 2) was obtained for the core Foetidae, including

4 Journal of Biogeography ª 2015 John Wiley & Sons Ltd 192 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction of Carex maritima Bipolar disjunction in Carex maritima

C. canescens ROM C. paniculata GRC C. remota YUG 0.96 Sect. Remotae 1 C. vernacula YUK ORE 100 1 C. vernacula Sect. Foetidae 100 C. vernacula CAL 1 C. stenophylla RU TAJ 100 C. stenophylla Sect. Divisae 86 C. stenophylla TCS C. sajanensis CHI_1 1 CHI_2 92 C. sajanensis C. sajanensis CHI_3 C. sajanensis NEP_1 0.98 77 C. sajanensis EHM_1 63 0.98 C. pseudofoetida TAJ_1 C. pseudofoetida TAJ_2 C. maritima ASK_1 C. maritima YUK_1a C. maritima YUK_1b C. maritima YUK_2 C. maritima YUK_3 C. maritima NWT_1 1 C. maritima NWT_2 100 C. maritima NWT_3 C. maritima NWT_4 C. maritima NWT_5 C. maritima SAS_1 C. maritima NUN_1a C. maritima NUN_1b C. maritima NUN_2 C. maritima MAN_1 C. maritima MAN_2 C. maritima NFL_1 Figure 2 Majority rule (50%) consensus C. maritima NFL_2 C. maritima GNL_1 tree derived from the Bayesian analysis of 0.99 C. maritima GNL_2 Carex maritima and the related species in 97 C. maritima GNL_3 C. maritima GNL_4 Sect. section Foetidae inferred from the combined C. maritima GNL_5 C. maritima ICE_1 Foetidae nuclear (ITS and ETS) and chloroplast ICE_2a 0 C. maritima (Core (5 trnK and rps16) matrix; C. remota, C. C. maritima ICE_3 C. maritima ICE_4 Foetidae) canescens and C. paniculata were used as C. maritima ICE_5 outgroups. Numbers above and below the C. maritima WSB_1 C. maritima SWI_1 branches represent the Bayesian posterior C. maritima SWI_2 probability (> 0.9 PP) and bootstrap C. maritima NOR_1 C. maritima NOR_2 (> 60% BS) values of the maximum C. maritima NOR_3 C. maritima NOR_4 likelihood analysis, respectively. A grey C. maritima RUE_1 rectangle highlights the C. maritima samples C. maritima RUW_1 C. maritima RUC_1 of the Southern Hemisphere. Vertical bars C. maritima ICE_2b RUW_2 indicate supraspecific taxa from the same 0.90 C. maritima 75 C. incurviformis COL_1 taxonomic group. Abbreviations after the C. incurviformis COL_2 C. maritima CLN_1 names correspond to the geographical 65 C. maritima CLN_2 regions of the world (Brummitt, 2001) and 0.99 C. maritima CLS_1 93 C. martitima AGS_1a to the population number. The scale bar C. maritima AGS_1b indicates substitutions per site. 0.0030 C. maritima AGS_3 all sampled section Foetidae species except C. vernacula. Nev- were found within the sampled C. maritima populations. ertheless, several species from section Foetidae, not sampled in Two of them were widely distributed but geographically the current study, could potentially also be part of the core overlapping in part: one of them was shared by 10 samples Foetidae. A strongly supported (100% BS/1 PP) monophyletic of C. maritima from Russia, Canada and Greenland (H1), C. stenophylla (sect. Divisae) was sister to the core Foetidae and the other by 26 samples of C. maritima from northern (100% BS/1 PP). Within the latter, C. sajanensis was retrieved North America and Europe (H2). In addition, H1 was also as monophyletic (92% BS/1 PP) and sister to a strongly sup- shared with one population of C. incurviformis and two of C. ported clade (97% BS/0.99 PP), including C. pseudofoetida, C. pseudofoetida. Interestingly, all C. maritima samples from incurviformis and C. maritima. Two different subclades were South America (5) shared the same exclusive haplotype detected: (1) a strongly supported lineage comprising all C. (H3). We found unique haplotypes for C. maritima samples maritima samples from South America (93% BS/0.99 PP); and from Norway (H4; one sample) and for samples from eastern (2) C. incurviformis (75% BS/0.90 PP). Russia (H5; two samples). Finally, C. sajanensis displayed two haplotypes (H5–H6) separated by four mutational steps from C. maritima haplotypes. Haplotype network

The cpDNA haplotype network obtained for the core Foeti- Estimation of divergence times dae (Fig. 3) revealed seven haplotypes and five missing hapl- otypes. A probability of 81% that all haplotypes have been The dating analyses produced a congruent topology with sampled is given by Dixon’s (2006) method. Five haplotypes respect to BI and ML analyses presented above (Fig. 4,

Journal of Biogeography 5 ª 2015 John Wiley & Sons Ltd 193 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction of Carex maritima T. Villaverde et al.

C. marima C. pseudofoeda (2) (1) India Norway H4 C. incurviformis (1) Colorado C. marima (10) North and Central Russia, Northwest C. marima (5) H1 Territories & Greenland South America H3 C. marima (26) C. marima (2) Alaska, Canada & Eastern Russia Europe H5 H2 Figure 3 Haplotype network of concatenated cpDNA sequences of Carex maritima, C. pseudofoetida, C. incurviformis and C. sajanensis. Circles represent the seven haplotypes found (H1–H7), lines represent single mutational steps, and small black C. sajanensis C. sajanensis circles represent missing haplotypes. H7 (1) H6 (4) Numbers of samples per haplotype are indicated in parentheses.

Table 1). The divergence time of the clade comprising core tures during the coldest month than populations from the Foetidae was 2.85 Ma (95% HPD: 0.93–5.01 Ma), which falls Southern Hemisphere (Table 2, Fig. 6). The PCA of the full in the late Pliocene to middle Pleistocene. The diversification climatic data set showed that PC1 explained 65.9% of the of the clade consisting of C. maritima, C. incurviformis and variance whereas PC2 and PC3 explained 14.7% and 9.4%, C. pseudofoetida could have occurred during the middle–late respectively (Appendix S1). A clear separation between Pliocene to early–middle Pleistocene (1.61 Ma; 95% HPD: Northern and Southern Hemisphere samples of C. maritima 0.61–2.96 Ma). Finally, the divergence of the clade consisting is also obtained when plotting PC1 and PC3 or PC2 and of C. maritima samples from South America could have PC3 (Appendix S2). begun during the middle–late Pleistocene (0.23 Ma; 95% HPD: 0.03–0.51 Ma). While the age of the core Foetidae Past and present distribution under climatic change could experience some variation after including some of the scenarios missing species from section Foetidae, the estimated age for C. maritima clade (the clade including all C. maritima sam- Current conditions ples) is reliable and should experience little or no variation after including unsampled species even if one or several of Our results show that the modelled ecological niche of C. those fell nested within the C. maritima clade. maritima, including Northern and Southern Hemisphere samples, predicts suitable areas in both hemispheres. Values for AUC were all above 0.9, which indicate a good fit of the Climatic environment models. The average AUC values for each group and the The phylogenetic PCA of the reduced climatic data set most important environmental variables detected in each showed that PC1 explained 50.35% of the variance whereas analysis are reported in Appendix S1. Scatter diagrams of the PC2 explained 22.29% (see Fig. 5). The variables with the variables used in Maxent analyses also depicted clear differ- highest loadings in PC1 were temperature seasonality ences between Northern and Southern Hemisphere popula- (BIO4), temperature range (BIO7) and minimum tempera- tions of C. maritima (Appendix S2). The modelled ecological ture of the coldest month (BIO6; see Appendix S1). Maxi- niche of C. maritima including only the Northern Hemi- mum and minimum values for each variable are shown by sphere populations predicts suitable habitats also in the groups (Table 2). Similar results were obtained when the Southern Hemisphere (Appendix S2). By contrast, the mod- analysis is not corrected with the phylogeny (results not elled ecological niche of C. maritima including only Southern shown). Northern and Southern Hemisphere samples of C. Hemisphere populations does not predict suitable habitats in maritima were clearly separated into two groups, probably the Northern Hemisphere. revealing some degree of ecological differentiation. The box- plots of the variables with the highest loadings revealed that Past conditions C. maritima populations from the Northern Hemisphere occur in localities with greater temperature oscillations The projection of suitable environments to past conditions through the year and a wider range of minimum tempera- in all lineages revealed a wider distribution range in LGM

6 Journal of Biogeography ª 2015 John Wiley & Sons Ltd 194 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction of Carex maritima Bipolar disjunction in Carex maritima

C. paniculata

C. canescens

C. remota 0.81 8.05 1 C. vernacula 1.80

1 Figure 4 Maximum credibility clade C. stenophylla phylogeny of the Bayesian divergence time 0.54 analysis considering Carex maritima and 0.83 1 C. sajanensis other related species in section Foetidae 9.04 0.63 carried out on a combined matrix of 0 nuclear (ITS and ETS) and plastid (5 trnK 1 C. incurviformis and rps16) sequences. Carex remota, C. 2.85 canescens, C. paniculata and C. stenophylla C. maritima were used as outgroups. Node bars 0.87 1 Southern Hemisphere represent the 95% highest posterior density 0.09 1.61 intervals of the divergence time estimates C. maritima 0.76 Northern Hemisphere + linked to nodes with posterior probabilities 0.23 C. pseudofoetida above 0.75 (values above branches) with MIOCENE PLIOCENE PLEISTOCENE mean ages inferred for clades in million years (below branches). 15.0 12.5 10.0 7.5 5.0 2.5 0.0 Ma scenarios than at the present time and in the LIG scenarios analysis during the middle–late Pliocene and early–middle (Appendix S2). Although suitable environments are predicted Pleistocene (Table 1, Fig. 4). in the Southern Hemisphere for C. maritima populations The other five bipolar Carex species manifest most of their from the Northern Hemisphere, when Southern Hemisphere distribution in the Northern Hemisphere (Govaerts et al., populations are analysed alone these areas are considerably 2014). At least C. arctogena, C. macloviana and C. maritima reduced in LGM scenarios and absent in the LIG scenario display a higher morphological variation in North America (Appendix S2). These results have to be interpreted with than in South America (Moore & Chater, 1971), which could caution due to the absence of fossils to validate the model. also support the idea that the bipolar species generally migrated southwards (Raven, 1963; Moore & Chater, 1971). Studies of other bipolar taxa have also suggested a north-to- DISCUSSION south dispersal as the most plausible migration direction (e.g. Moore & Chater, 1971; Vollan et al., 2006; Popp et al., Pleistocene north-to-south long-distance dispersal 2011). The C. maritima biogeographical history elucidated in Haplotypes H1 and H2 are widely distributed throughout our study appears to be congruent with the predominantly the Northern Hemisphere (North America, Europe and Asia) inferred pattern, and it seems that Northern Hemisphere to and comprise the highest number of haplotype connections South Hemisphere dispersal is predominant in plant dispers- (Fig. 3), implying under the coalescent theory that they als (reviewed in Wen & Ickert-Bond, 2009). However, other amount to the ancestral haplotypes (Posada & Crandall, plant genera present the opposite direction of dispersal. For 2001). South American C. maritima populations are mono- example, the centre of origin of the genus Larrea (Zygophyll- phyletic and nested with a strong statistical support within aceae) is located in South America and this genus was the Northern Hemisphere accessions (Figs 2 & 3). In addi- inferred to have migrated to North America during the late tion, the haploid genotype diversity pattern consisting of Neogene by long-distance dispersal, using way stations in four different haplotypes found in relation to the C. mariti- Peru and Bolivia and probably mediated by birds (Lia et al., ma populations of the Northern Hemisphere (H1, H2, H4, 2001). Likewise, the Rubiaceae family was inferred to have H5), whilst a single haplotype was detected for the southern migrated from South to North America during the late Pal- populations (H3; Fig. 3), suggests a migration event from aeocene–early Eocene using land bridges (Antonelli et al., the Northern Hemisphere to the Southern Hemisphere as 2009), as was Hoffmannseggia glauca (Fabaceae) via birds the most plausible explanation. This evidence, together with during the late Miocene or later (Simpson et al., 2005). the fact that 11 out of the 15 species in Carex sect. Foetidae (eMonocot Cyperaceae; http://cyperaceae.e-monocot.org, Mountain-hopping or direct long-distance dispersal? accessed 3 December 2014) are also distributed in the North- ern Hemisphere, supports the hypothesis that C. maritima The subsequent question arises of how the inferred north- originated in the Northern Hemisphere, according to our to-south middle–late Pleistocene long-distance dispersal

Journal of Biogeography 7 ª 2015 John Wiley & Sons Ltd 195 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction of Carex maritima T. Villaverde et al.

Table 1 Divergence times of clades in Carex sect. Foetidae and outgroups presented as the posterior probability followed by the mean time to the most recent common ancestor in million years and the 95% highest posterior density (HPD) interval obtained from the divergence time analysis of the combined nuclear (ITS and ETS) and plastid (50trnK intron and rps16) regions. Carex maritima populations of the Northern Hemisphere (NH) and Southern Hemisphere (SH), respectively, are indicated.

Posterior 95% HPD Clade probability Mean (Ma) interval (Ma)

C. remota + C. vernacula 0.81 8.0522 2.7638 13.1959 C. vernacula 1 1.8038 0.2665 3.7436 C. stenophylla + Core Foetidae 0.83 9.0452 3.6505 14.2733 (C. sajanensis + C. incurviformis + C. maritima + C. pseudofoetida) C. stenophylla 1 0.5365 0.0291 1.2872 Core Foetidae 1 2.8456 0.9327 5.0077 (C. sajanensis + C. incurviformis + C. maritima + C. pseudofoetida) C. sajanensis 1 0.6275 0.0440 1.4295 C. incurviformis + C. maritima NH + C. maritima SH + C. pseudofoetida 1 1.6110 0.6062 2.9603 C. incurviformis 0.87 0.0884 0 0.3099 C. maritima SH 0.76 0.2266 0.0286 0.5092

have migrated stepwise by mountain-hopping all the way C. maritima Northern Hemisphere C. maritima Southern Hemisphere through the Andes, with a posterior extinction of most of C. incurviformis

46 the intermediate populations; or (2) Northern Hemisphere C. pseudofoetida C. sajanensis populations could have been disseminated by a direct long- distance dispersal to South America, where they subsequently

PC2 colonized northwards or southwards until reaching their cur- rent distribution (Fig. 1). The mountain-hopping hypothesis (Ball, 1990) proposes a long-distance, stepwise migration of arctic and temperate -2 0 2 taxa using mountains peaks as stepping-stones to cross the -2 0 2 4 6 tropics. A route connecting North and South America PC1 through the American cordillera has been in place since the late Miocene epoch (Smith, 1986). Then, a gradual uplift of Figure 5 Scatter plot of the first two components explaining up the cordillera during the late Pliocene created the high to 72.64% of the observed variance, derived from the principal mountainous environment with a much colder climate later components analysis as corrected by phylogeny and depicting on during the Pleistocene compared to that occurring today the position in a climate-niche space of Northern and Southern at the same latitudes and elevations (van der Hammen, Hemisphere samples of Carex maritima (black and white circles, respectively), C. pseudofoetida (triangles), C. incurviformis 1974). According to our results, from the Last Interglacial (diamonds) and C. sajanensis (crosses). (LIG; c. 120 ka) to the present time similar ecological niches could have existed that were suitable for C. maritima in South America (although these results should to be taken Table 2 Maximum and minimum values of the variables with with caution, see Results; Appendix S2). Therefore, we can- the highest loadings for principal component 1 for the Northern Hemisphere populations (NH) and Southern Hemisphere not rule out the mountain-hopping migration since the early populations (SH) of Carex maritima. Bioclimatic variables Pleistocene, with a subsequent extinction of most of the correspond to temperature seasonality (BIO4, SD), the northern South American intermediate populations. In addi- temperature range (BIO7 = BIO6 – BIO5, C), the minimum tion, the sister relationship between C. maritima of the temperature during the coldest month (BIO6, C) and the Southern Hemisphere and C. incurviformis (present in wes- maximum temperature during the coldest month (BIO5). tern North America yet reaching southern latitudes) could BIO4 BIO7 BIO6 also support this hypothesis (nonetheless see lack of clade support; Fig. 2). As already demonstrated by Heide (2002), C. maritima HN min. 35.33 14.2 44.9 changes in flowering requirements would not have been nec- max. 177.28 58 3.5 essary for the other bipolar Carex species to migrate across C. maritima SH min. 24 15.4 9.6 max. 32.78 25.4 0.6 of the tropical belt, still making mountain-hopping a plausi- ble premise. Alternatively, C. maritima could have reached the South- could have occurred. According to our data, the current C. ern Cone by a direct long-distance dispersal event, with a maritima distribution can be explained by either of two subsequent genetic differentiation in the South American hypotheses: (1) Northern Hemisphere populations could continent and a northward or southward colonization along

8 Journal of Biogeography ª 2015 John Wiley & Sons Ltd 196 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction of Carex maritima Bipolar disjunction in Carex maritima

(a) (b)

Figure 6 Carex maritima boxplots comparing the three bioclimatic variables with the highest loadings regarding the first

component of the bioclimatic principal BIO4 (SD) components analysis (PCA) taking into BIO7 (°C) account the Northern and Southern Hemisphere samples (NH and SH). (a) Temperature seasonality (BIO4), (b) annual NH SH NH SH temperature range (BIO7, i.e. minimum (c) temperature during the warmest month minus maximum temperature during the coldest month, BIO6 – BIO5), and (c) minimum temperature during the warmest month (BIO6). Each box represents the

interquartile range which contains 50% of BIO6(°C x 10) the values and the median (horizontal line across the box); the whiskers are the lines that extend from the box to the highest and lowest values, excluding outliers (o). NH SH the Andes, as suggested for the bipolar C. arctogena (Villav- long-distance dispersal, unlike other bipolar species (C. erde et al., 2015). The molecular data in this study, without microglochin; Savile, 1972). On the one hand, species with genetic structure between northern and southern populations small seeds and from water-influenced habitats are often within each hemisphere, but strong genetic structure between highly dispersible taxa (McGlone et al., 2001). On the other both hemispheres, could support the direct long-distance dis- hand, the long-distance dispersal of seeds might not neces- persal hypothesis and subsequent genetic differentiation. sarily be driven by standard dispersal vectors inferred from Nevertheless, more information is still needed to confirm plant morphology (as described by Higgins et al., 2003) or either the mountain-hopping or the direct long-distance dis- by regular events; in fact, great long-distance dispersals persal hypothesis. (> 100 km) are usually associated with stochastic events (unusual behaviour of regular events or a combination of vectors; Nathan et al., 2008). Thus, arctic species have been Breeding system, dispersal syndrome and the bipolar demonstrated to migrate enormous distances despite the lack disjunction of specific syndromes (Abbott & Brochmann, 2003). Some self-fertilization is a reproductive characteristic dis- Considering the extreme dispersal distance, together with played by many species with disjunct populations in the tem- the shape and structure of the C. maritima propagules, we perate zones (Carlquist, 1983). This attribute could favour consider that its dispersal was more likely to have been med- local survival and establishment after long-distance dispersal iated by migratory animals than by wind or ocean currents, events, given that a single propagule of self-compatible which seem insufficient for such an enormous task. Some individuals could in principle be sufficient to start a sexually birds which migrate from North America to temperate zones reproducing colony (Baker, 1955). In congruence, Carex spe- of South America have already been pointed out as the most cies are predominantly monoecious and in general highly likely dispersal agents of the several disjunct plant groups self-pollinated (Friedman & Barrett, 2009), which has been (Cruden, 1966, and references therein; Popp et al., 2011). inferred from studies based on hand pollinations, isozyme Moreover, arrivals of Carex species to newly formed islands work (e.g. Ohkawa et al., 2000; Friedman & Barrett, 2009) have predominantly been reported to happen when seeds and microsatellite data (e.g. Escudero et al., 2010b, 2013). were embedded in mud attached to birds’ feet or else when This characteristic could explain, at least in part, the often eaten and carried inside by birds (Carlquist, 1967). There- successful colonization of Carex species after a long-distance fore, we consider it plausible that C. maritima could have dispersal event (Moore & Chater, 1971; Ball, 1990; Escudero acquired its bipolar distribution by means of bird-mediated et al., 2009). dispersal. Carex maritima inhabits water-influenced areas (e.g. lakes, river, ocean shores or snowmelt water areas) or else popu- CONCLUSIONS lates hydromorphic soils (e.g. beaches, fens, alluviums), and seed dispersal of C. maritima could be mediated by birds, This study contributes to the general knowledge regarding wind or ocean currents. Except for their small size, fruits of biogeographical patterns of bipolar taxa whilst presenting a C. maritima lack the evident morphological features for a combination of multiple approaches (phylogenetic and

Journal of Biogeography 9 ª 2015 John Wiley & Sons Ltd 197 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction of Carex maritima T. Villaverde et al. phylogeographical analyses, together with divergence time Carlquist, S. (1967) The biota of long-distance dispersal. V. estimates and bioclimatic data) to test the traditional Plant dispersal to Pacific Islands. Bulletin of the Torrey hypotheses used to understand the distribution of bipolar Botanical Club, 94, 129–162. species. Carex maritima populations of the Southern Hemi- Carlquist, S. (1983) Intercontinental dispersal. Sonderbd sphere were retrieved as a monophyletic lineage within a naturwiss Vereins in Hamburg, 7,37–47. paraphyletic C. maritima. The phylogeographical structure Clement, M., Posada, D. & Crandall, K. (2000) TCS: a com- found within C. maritima suggests that the bipolar disjunc- puter program to estimate gene genealogies. Molecular tion could be explained by a middle–late Pleistocene long- Ecology, 9, 1657–1660. distance dispersal derived from the Northern Hemisphere. Crisp, M.D., Trewick, S.A. & Cook, L.G. (2011) Hypothesis Our study highlights the importance of long-distance dis- testing in biogeography. Trends in Ecology and Evolution, persal mechanisms to explain this fascinating plant distribu- 26,66–72. tion pattern. Cruden, R.W. (1966) Birds as agents of long-distance dis- persal for disjunct plant groups of the temperate Western Hemisphere. Evolution, 20, 517–532. ACKNOWLEDGEMENTS Darwin, C. (1872) The origin of the species. Murray, London. The authors thank all staff from herbaria CAN, COLO, E, Degnan, J.H. & Rosenberg, N.A. (2009) Gene tree discor- M, MSB, SI, UPOS and WIN for granting us access to their dance, phylogenetic and the multispecies coalescent. collections and for providing plant material; E. Maguilla Trends in Ecology and Evolution, 24, 332–340. (Universidad Pablo de Olavide, UPO) and Francisco Dixon, C. (2006) A means of estimating the completeness of Rodrıguez-Sanchez (Estacion Biologica de Donana,~ EBD- haplotype sampling using the Stirling probability distribu- CSIC) for their help with the Maxent analyses and map tion. Molecular Ecology Notes, 6, 650–652. editing; L.P. Bruederle (University of Colorado, Denver) and Donoghue, M. (2011) Bipolar biogeography. Proceedings of P. Vargas (Real Jardın Botanico de Madrid) for assistance in the National Academy of Sciences USA, 108, 6341–6342. plant collections; and M. Mıguez and F.J. Fernandez (UPOS) Drummond, A.J. & Rambaut, A. (2007) BEAST: Bayesian for technical support. We also thank the University of evolutionary analysis by sampling trees. BMC Evolutionary Helsinki master’s student A. Ginter for translations of Biology, 7, 214. Russian data labels. This research was supported by the Drummond, A.J., Suchard, M.A., Xie, D. & Rambaut, A. 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Savile, D.B.O. (1972) Arctic adaptations in plants. Monograph for phylogenetics. PCR protocols: a guide to methods and No. 6. Canada Department of Agriculture Research applications (ed. by M. Innis, D. Gelfand, D. Sninsky and Branch, Ottawa. T. White), pp. 315–322. Academic Press, San Diego, CA. Scotese, C., Gahagan, L. & Larson, R. (1988) Plate tectonic reconstructions of the Cretaceous and Cenozoic ocean basins. Tectonophysics, 155,27–48. SUPPORTING INFORMATION Scotland, R.W. (2011) What is parallelism? Evolution and Additional Supporting Information may be found in the 13 – Development, , 214 227. online version of this article: Shaw, J., Lickey, E.B., Beck, J.T., Farmer, S.B., Liu, W., Miller, J., Siripun, K.C., Winder, C.T., Schilling, E.E. & Appendix S1 Supplementary tables. Studied material of Ca- Small, R.L. (2005) The tortoise and the hare II: relative rex maritima, related species and outgroups (Table S1); utility of 21 noncoding chloroplast DNA sequences for molecular characteristics of the amplified regions (Table S2); phylogenetic analysis. American Journal of Botany, 92, results from the principal components analysis of 19 biocli- 142–166. matic variables from the WorldClim database, uncorrected Simpson, B.B., Tate, J.A. & Weeks, A. (2005) The biogeogra- (on full and reduced data sets) and corrected for phylogeny – phy of Hoffmannseggia (Leguminosae, Caesalpinioideae, (Tables S3 S5); description of bioclimatic variables (Table Maxent Caesalpinieae): a tale of many travels. Journal of Biogeogra- S6); and AUC values from analyses (Table S7). phy, 32,15–27. Appendix S2 Supplementary figures. Distribution map of Smith, J.M.B. (1986) Origins of the Australasian tropicalpine Carex maritima (Fig. S1); ecological niche models of C. floras. CSIRO, Melbourne. maritima groups fitted to current climatic conditions from Maxent Stamatakis, A. (2006) RAxML-VI-HPC: maximum likeli- analyses (Fig. S2), and projections of the models to – hood-based phylogenetic analyses with thousands of taxa Last Glacial Maximum (18 21 ka; MIROC and CCSM mod- – and mixed models. Bioinformatics, 22, 2688–2690. els) and Last Interglacial Period (c. 120 140 ka) (Fig. S3); Starr, J.R., Harris, S.A. & Simpson, D.A. (2003) Potential of scatter plots of the uncorrelated bioclimatic variables used in Maxent the 5 and 3 ends of intergenic spacer (IGS) of rDNA in analyses (Fig. S4); and scatter plot of the three first the Cyperaceae: new sequences for lower-level phylogenies components from the principal components analysis of the in sedges with an example from Uncinia Pers. International full data set (Fig. S5). Journal of Plant Sciences, 164, 213–227. Villaverde, T., Escudero, M., Martın-Bravo, S., Bruederle, BIOSKETCH P.L., Luceno,~ M. & Starr, J.R. (2015) Direct long-distance dispersal best explains the bipolar distribution of Carex Tamara Villaverde is a PhD student at Pablo de Olavide arctogena (Carex sect. Capituligerae, Cyperaceae). Journal University, Seville (Spain). Her research is focused on the of Biogeography, doi:10.1111/jbi.12521. evolution and phylogeography of angiosperms, with special Vollan, K., Heide, O.M., Lye, K.A. & Heun, M. (2006) interest in the systematics and biogeography of the genus Genetic variation, taxonomy and mountain-hopping of Carex (Cyperaceae). four bipolar Carex species (Cyperaceae) analysed by AFLP Author contributions: M.E. and M.L. conceived the idea; 54 – fingerprinting. Australian Journal of Botany, , 305 313. T.V., S.M-B and M.L. collected the plant material; T.V., M.E. Wen, J. & Ickert-Bond, S.M. (2009) Evolution of the Madre- and S.M-B, carried out the lab work and analysed the data; an-Tethyan disjunctions and the North and South Ameri- T.V., M.E. and S.M-B led the writing and drafted the manu- can amphitropical disjunctions in plants. Journal of script, although all authors contributed to its preparation. Systematics and Evolution, 47, 331–348. White, T.J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplifica- tion and direct sequencing of fungal ribosomal RNA genes Editor: Liliana Katinas

12 Journal of Biogeography ª 2015 John Wiley & Sons Ltd 200 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

Journal of Biogeography

SUPPORTING INFORMATION

Long-distance dispersal during the middle–late Pleistocene explains the bipolar disjunction of Carex maritima (Cyperaceae)

Tamara Villaverde, Marcial Escudero, Modesto Luceño and Santiago Martín-Bravo

Appendix S1 Supplementary tables. Studied material of Carex maritima, related species and outgroups (Table S1); molecular characteristics of the amplified regions (Table S2); results from the principal components analysis of 19 bioclimatic variables from the WorldClim database, uncorrected (on full and reduced data sets) and corrected for phylogeny (Tables S3–S5); description of bioclimatic variables (Table S6); and AUC values from MAXENT analyses (Table S7).

201 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

Table S1 List of material studied of Carex maritima, C. incurviformis, C. pseudofoetida, C. sajanensis, C. vernacula and the outgroups C. stenophylla, C. canescens, C. remota and C. paniculata, including population code, coordinates, voucher information, corresponding clade and GenBank accessions for markers used for molecular studies. Population codes correspond to geographical regions of the world (Brummitt, 2001) and population number.

Species Pop. code Latitude Longitude Label information GenBank accession ETS/ITS/rps16/trnK C. paniculata GRC 21.22555 39.78673 Greece, Epiro, Ioannina, Kambos Despoti. KR827052/ KR827095 / subsp. paniculata M. Luceño 0808ML, 23/VI/2008. KR827140 / KR827191 (UPOS3419) C. canescens ROM 45.60321 24.61613 Romania, Carpathians, Bâle Lake. M. KR827051/ KR827094 / Pusças s.n., 20/07/2013. (Personal KR827139 / KR827190 collection) C. remota YUG 19.09025 43.15227 Montenegro, High Dinarics, Durmitor KR827053/ KR827096 / National Park. P. Jiménez-Mejías KR827141 / KR827192 198PJM10, 17/VII/2010. (UPOS4006) C. vernacula CAL 38.44445 -119.32149 USA, California, Wheeler Peak. Bell 1459. -/EU001077/-/- (WS s.n.) C. vernacula YUK 61.28698 -138.52892 Canada, Yukon. Kluane Lake. L. P. -/ KR827099 / KR827144 /- Bruederle 08142010_01c. 14/VIII/2010. (COLO s.n.) C. vernacula ORE - - USA, Oregon. Mason 9130. (POM s.n.) /AF285022/-/-

202 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

C. stenophylla TCS 40.42275 44.23447 Armenia, Aragatsotn Province, Mt. Aragats. KR827054/ KR827098 / G. Fayuush 07-1385, 23/VI/2007. (NY s.n.) KR827143 / KR827194

C. stenophylla TAJ 38.68333 73.71667 Tajikistan, Gorno-Badakhshan, East Pamir. -/ KR827097 / KR827142 / B. Dickoré 18239, 16/IX/2002. KR827193 (MSB162331) C. stenophylla RU - - Russia. (MO04981469) EU001224/EU001070/-/- C. sajanensis CHI_1 30.30000 90.60000 China, Xizang Zizhiqu, Dangxiong Xian. B. -/-/ KR827145 / KR827195 Dickoré 3765, 11/VIII/1989. (MSB140868)

C. sajanensis CHI_2 33.55000 91.35000 China, Qinghai, Tibet. B. Dickoré 4409, -/-/ KR827146 / KR827196 24/VIII/1989. (MSB140876) C. sajanensis CHI_3 30.30000 90.60000 China, Xizang Zizhiqu, Dangxiong Xian. B. KR827055/-/ KR827147 /- Dickoré 4010, 14/VIII/1989. (MSB142360)

C. sajanensis EHM_1 27.93333 88.63333 India, Sikkim, Chuu Valey. Edinburgh KR827056/ KR827100 / Expedition to Northern Sikkim (1996) 364, KR827148 / KR827197 20/VII/1996. (E00047590) C. sajanensis NEP_1 27.90611 86.70556 Nepal, Sagarmatha, Machhermo Kola. First KR827057/ KR827101 / Darwin Nepal Fieldwork Training KR827149 / KR827198 Expedition 171. 15/V/2004. (E00229251) C. pseudofoetida TAJ_1 38.48333 74.31667 Tajikistan, Gorno-Badakhshan, Murgab. B. -/ KR827102 / KR827151 / Dickoré 18037, 12/IX/2009. (MSB162329) KR827200

C. pseudofoetida TAJ_2 38.61667 72.86667 Tajikistan, Gorno-Badakhshan, East Pamir. -/-/ KR827150 / KR827199 B. Dickoré 17842, 09/IX/2002. (MSB162332)

203 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

C. incurviformis COL_1 38.86297 -105.37929 USA, Colorado, Park County. D. Randolph KR827058/ KR827103 / 17402, 23/VII/1984. (CAN499701) KR827152 / KR827201

C. incurviformis COL_2 39.19275 -105.45410 USA, Colorado, Park County, Pike National -/DQ115186/-/- Forest, Horseshoe Cirque area. Tallent 517. (MICH s.n.) C. maritima AGS_1a -53.34666 -68.27933 Argentina, Tierra del Fuego, Río Grande. S. KR827073/-/ KR827167 / Martín-Bravo 42SMB10, 15/I/2010. KR827218 (UPOS4277) C. maritima AGS_1b -53.34667 -68.27930 Argentina, Tierra del Fuego, Río Grande. S. KR827076/ KR827120 / Martín-Bravo 42SMB10, 15/I/2010. KR827170 / KR827220 (UPOS4274) C. maritima AGS_2 -54.05803 -67.39000 Argentina, Tierra del Fuego, Río Grande. J. KR827077/ KR827121 / Starr P9-1 10013. 12/I/2010 (UPOS3930) KR827171 / KR827221

C. maritima CLN_1 -28.61667 -69.86667 Chile, Atacama, El Tránsito. S. Teillier 4953, KR827059/ KR827104 / 15/II/2002. (MIN934748) KR827153 / KR827202

C. maritima CLN_2 -20.98333 -68.55000 Chile, Iquique, Pica. S. Teillier 3258, KR827061/ KR827106 /-/ 24/I/1994. (MIN934751) KR827204 C. maritima CLS_1 -52.76021 -69.02174 Chile, Región Magallanes-Antártica, Tierra KR827066/ KR827112 / del Fuego. M. Luceño 18ML06, 6/I/2006. KR827158 / KR827210 (UPOS1830) C. maritima ASK_1 68.19472 -152.74750 USA, Alaska, Chandler Lake. B. A. Benett -/ KR827113 / KR827160 / 02-393, 23/VII/2002. (CAN589262) KR827212

C. maritima GNL_1 65.45000 -52.55000 Greenland, Kangerdluarssuk, Qivaqe. S. KR827062/ KR827107 /-/ Holt 785, 15/VIII/1977. (CAN488203) KR827205

204 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

C. maritima GNL_2 73.20404 -24.49918 Greenland, Ymer Island, Botanikerbugten. KR827067/-/ KR827159 / T. Sørensen 3087, 17/VIII/1932. KR827211 (CAN17509) C. maritima GNL_3 72.86333 -25.13333 Greenland, Ella Island, Cape Oswald. T. KR827069/-/ KR827162 / - Sørensen 316, 24/VII/1932. (CAN17503) C. maritima GNL_4 61.00098 -46.44850 Greenland, Qaleragdlit fjord. M. Luceño KR827074/ KR827119 / 4707ML, 5/VIII/2007. (UPOS4484) KR827168 / KR827219 C. maritima GNL_5 61.16096 -45.41469 Greenland, Narsarsuaq, Kieqtotsermiat KR827086/ KR827130 / glacier. M. Luceño 9307ML, 16/VIII/2007. KR827181 / KR827229 (UPOS4512) C. maritima ICE_1 64.07082 -16.97465 Iceland, Skaftafell N.P. M. Guzmán s.n., -/EU541874/-/- 15/Vlll/2005. (UPOS706) C. maritima ICE_2a 65.01748 -19.21685 Iceland, My vatnn. G. Kaule s.n., KR827084/-/ KR827179 / 18/VIII/1970. (M0177749) KR827227 C. maritime ICE_2b 65.01748 -19.21685 Iceland, My vatnn. G. Kaule s.n., -/-/KR869806/- 9/VIII/1970. (M0177749) C. maritima ICE_3 65.81401 -16.37628 Iceland, Dettifoss. M. Guzmán s.n., KR827090/ KR827134 / 22/VIII/2005. (UPOS00707) KR827184 / KR827233 C. maritima ICE_4 63.55043 -19.34967 Iceland, Vik, Drangshliðardalur. M. Luceño KR827093/ KR827138 / 4706ML, 7/VIII/2006. (UPOS1957) KR827187 / KR827236 C. maritima ICE_5 64.39412 -16.78246 Iceland, Vatnajökull. M. Luceño 5206ML, -/-/ KR827188 /- 8/VIII/2006. (UPOS1963) C. maritima NFL_1 47.38583 -54.69931 Canada, Newfoundland and Labrador, KR827079/ KR827123 / Burnt Island. B. S. Hay 87329, 25/VII/1987. KR827173 / KR827223 (CAN545627) C. maritima NFL_2 47.61587 -58.86786 Canada, Newfoundland and Labrador, KR827080/ KR827125 / Pointe Riche Peninsula. A. Bouchard 91022, KR827175 /- 30/VII/1991. (CAN564151)

205 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

C. maritima MAN_1 58.39004 -94.36161 Canada, Manitoba, Churchill. H. -/ KR827136 / KR827185 / Doppelbaur 134, 29/VII/1965. (M0177754) KR827235

C. maritima MAN_2 57.84694 -92.76806 Canada, Manitoba, Wapusk National Park. -/DQ115214/-/- E. Punter (03-711), 21/VII/2003. (CAN591811) C. maritima NOR_1 70.23601 24.94370 Norway, Lapland, Stabbursnes. M. Luceño KR827068/ KR827114 / 7305ML, 8/VIII/2005. (UPOS00370) KR827161 / KR827213

C. maritima NOR_2 68.19203 13.70330 Norway, Lofoten Islands. M. Escudero KR827071/ KR827116 / 44ME09. 27/VII/2009. (UPOS s.n.) KR827164 / KR827215 C. maritima NOR_3 71.02708 -8.39625 Norway, Havhestberget, Jan Mayen Island. KR827072/ KR827117 / J. Lid s.n., 10/VIII/1930. (CAN281858) KR827165 / KR827216

C. maritima NOR_4 78.21307 15.66346 Norway, Longyearbyen, Spitzbergen. F. -/-/ KR827189 / KR827237 Hörl s.n., 14/VIII/1961. (M0177748) C. maritima NUN_1a 63.73333 -68.45000 Canada, Nunavut, Baffin Island. S. G. Aiken KR827078/ KR827122 / 86-431, 19/VIII/1986. (CAN518311) KR827172 / KR827222 C. maritima NUN_1b 63.73333 -68.45000 Canada, Nunavut, Baffin Island. S. G. Aiken KR827085/ KR827129 / 86-346, 16/VIII/1986. (CAN518226) KR827180 / KR827228 C. maritima NUN_2 64.31868 -96.01683 Canada, Nunavut, Baker Lake. S. A. Edlund KR827091/ KR827135 /-/ 350, 31/VII/1983. (CAN495360) KR827234 C. maritima NWT_1 79.89869 -90.97104 Canada, Northwest Territories, Axel -/ KR827108 / KR827155 / Heiberg Island. A. E. Porsild 18643, KR827206 1/VIII/1953. (CAN223325) C. maritima NWT_2 64.63998 -84.11801 Canada, Northwest Territories, KR827063/ KR827109 / Southampton Island. D. K. Brown s.n., KR827156 / KR827207 20/VII/1952. (CAN258613)

206 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

C. maritima NWT_3 79.15114 -75.17893 Canada, Northwest Territories, Ellesmere KR827064/ KR827110 /-/ Island. J. M. Gillett 18344A, 21/VII/1979. KR827208 (CAN454023) C. maritima NWT_4 69.06200 -105.10000 Canada, Northwest Territories, Victoria KR827070/ KR827115 / Island. L. J. Gillespie 1120, 26/VII/1997. KR827163 / KR827214 (CAN582314)

C. maritima NWT_5 72.00000 -125.00000 Canada, Northwest Territories, Banks KR827087/ KR827131 /-/ Island. J. M. Gillett 18833, 25/VII/1981. KR827230 (CAN464428) C. maritima RUC_1 57.62112 60.67165 Russia, Taymyr. Matveeva et al. s.n. , KR827060/ KR827105 / 15/VII/1965. (CAN327344) KR827154 / KR827203 C. maritima RUE_1 72.95000 121.66667 Russia, Sakha Republic. A. Tolmatsheur -/ KR827118 / KR827166 / s.n., 25/VII/1956. (CAN256245) KR827217 C. maritima RUW_1 66.28633 36.80000 Russia, Murmansk Oblast, Murmansk. E. KR827092/ KR827137 / Pobedimova, s.n., 29/VIII/1958. KR827186 /- (CAN377158) C. maritima RUW_2 68.48859 37.08493 Russia, Murmansk Oblast, Murmansk. E. KR827088/ KR827132 / Pobedimova s.n. 20/VIII/1958. (M0177753) KR827182 / KR827231

C. maritima SAS_1 59.46133 -109.81917 Canada, Saskatchewan, Lake Athabasca. G. KR827083/ KR827128 / W. Argus 8193, 28/VII/1972. (CAN351228). KR827178 / KR827226

C. maritima SWI_1 46.52271 9.88484 Switzerland, Engadine, Punt Muragl. J. KR827075/-/ KR827169 /- Höller s.n., 2/VIII/1965. (M0177741) C. maritima SWI_2 46.00715 7.74323 Switzerland, Zermatt, Trockener Steg. M. KR827082/ KR827127 / Luceño 25ML12 2/2, 11/VIII/2012. KR827177 / KR827225 (UPOS4997)

207 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

C. maritima WSB_1 70.67088 70.13672 Russia, Tyumen, Yamal Peninsula. O. KR827065/ KR827111 / Rebristaja s.n., 12/VIII/1983. (E00639424) KR827157 / KR827209 C. maritima YUK_1a 61.28698 -138.52892 Canada, Yukon. Kluane Lake. L. P. -/ KR827124 / KR827174 /- Bruederle 07142010 02. 14/VII/2010. (COLO s.n.) C. maritima YUK_1b 61.28698 -138.52892 Canada, Yukon. Kluane Lake. L. P. KR827081/ KR827126 / Bruederle 07142010 01. 14/VII/2010. KR827176 / KR827224 (COLO s.n.) C. maritima YUK_2 60.73167 -135.06500 Canada, Yukon, Kishwoot Island. B. A. KR827089/ KR827133 / Benett 06-033, 3/VII/2006. (CAN589263) KR827183 / KR827232 C. maritima YUK_3 - - Canada, Yukon. Waterway et al. 96.098 -/AY757421/-/- (MTMG s.n.) C. maritima Not 47.15778 10.58944 Austria, Tirol, Zams. M. Hellweger s.n. - included VI/1875. in molecular analyses C. maritima Not 58.29501 11.64168 Sweden, Bohuslän. Skee s.n., 19/VIII/1948. - included (UPOS s.n.) in molecular analyses C. maritima Not 46.50523 11.37101 Italia, Bolzano, Schlernhaus. Thomas et al. - included s.n., 19/VII/2006. (BOZ-PVASC9171) in molecular analyses

208 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

Table S2 Characteristics of the DNA regions sequenced for complete data sets including Carex maritima, related species in section Foetidae and outgroups.

Complete data set

5′trnKCarexF– rps16F–rpsR ITS1/ 5.8S/ ITS2 ETS1f – 18S-R 5′trnkCarexR Description Internal transcribed External transcribed intron of plastid region Intergenic spacer of spacers 1 and 2 and 5.8S spacer of ribosomal plastid region ribosomal RNA RNA

References Blattner (1999) and White Starr et al. (2003) Escudero & Luceño (2009) Shaw et al. (2005) (1990) Total number of sequences in the 52 45 48 52 alignment Aligned length (bp) 612 551 654 878

Ungapped length range 481-610 307-547 614-639 786-875

% Identical sites 67.30% 79.10% 88.70% 56.70%

% Pairwise identity 96.60% 97.90% 98.10% 92.30%

Variable characters 90 78 14 39

Parsimony-informative 57 38 4 9 characters Number of informative indels 0 0 2 2

Mean % G+C content 60.50% 53.10% 22.00% 24.20%

Substitution model GTR + I (ITS1) / HKY (5.8s) / HKY + I F81 + I GTR GTR + G (ITS2)

209 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

Table S3 Loadings matrix obtained by the principal components analysis uncorrected by phylogeny of 19 bioclimatic variables on Carex maritima, C. incurviformis, C. pseudofoetida and C. sajanensis using the reduced data set.

PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PC11 PC12 PC13 PC14 PC15 PC16 PC17 PC18 PC19 BIO1 -0.226 0.335 -0.067 0.101 0.049 0.099 0.034 -0.100 0.167 0.031 0.242 0.420 0.467 -0.369 0.272 0.257 0.200 -0.017 0.000

BIO2 0.121 0.310 -0.029 -0.484 -0.418 -0.248 -0.294 0.104 0.237 0.373 0.195 -0.002 -0.153 0.172 0.064 0.114 0.133 0.006 0.000

BIO3 -0.109 0.356 0.273 -0.209 -0.193 -0.424 0.154 -0.297 -0.464 -0.432 -0.017 -0.004 0.082 0.002 -0.020 -0.050 -0.050 0.011 0.000

BIO4 0.252 -0.191 -0.285 -0.002 -0.120 0.050 0.068 -0.031 -0.177 -0.261 0.094 0.000 -0.026 0.230 0.255 0.279 0.286 -0.640 0.000

BIO5 0.016 0.296 -0.458 0.082 -0.256 0.073 0.085 -0.201 0.044 0.097 -0.644 -0.174 -0.012 -0.194 -0.034 0.060 -0.030 -0.023 -0.278

BIO6 -0.272 0.219 0.117 0.162 0.156 0.004 0.089 -0.096 0.041 0.127 -0.244 -0.192 -0.236 0.197 0.099 0.297 0.223 0.010 0.664

BIO7 0.266 -0.091 -0.294 -0.123 -0.251 0.025 -0.052 0.011 -0.022 -0.082 -0.024 0.113 0.221 -0.266 -0.108 -0.259 -0.225 -0.018 0.695

BIO8 0.084 0.295 -0.393 0.050 0.467 -0.343 -0.050 0.499 -0.369 0.130 0.019 0.009 0.063 0.038 -0.025 0.005 -0.044 0.011 0.000

BIO9 -0.256 0.216 0.119 0.106 -0.322 0.416 -0.355 0.546 -0.123 -0.354 -0.077 -0.090 0.053 0.062 -0.025 -0.010 0.018 -0.010 0.000

BIO10 -0.058 0.280 -0.449 0.182 -0.059 0.208 0.178 -0.217 0.081 -0.212 0.486 -0.021 -0.266 0.296 -0.107 -0.159 -0.046 0.269 0.000

BIO11 -0.258 0.275 0.108 0.068 0.101 0.015 0.013 -0.057 0.169 0.185 0.083 -0.015 -0.048 0.009 -0.134 -0.400 -0.286 -0.705 0.000

BIO12 -0.293 -0.165 -0.130 -0.085 -0.073 -0.030 0.069 0.000 -0.101 0.149 -0.193 0.187 0.222 0.331 0.176 -0.576 0.467 0.087 0.000

BIO13 -0.268 -0.112 -0.153 -0.320 0.152 0.219 -0.247 -0.197 -0.341 0.108 0.218 -0.397 -0.187 -0.443 0.161 -0.089 0.142 -0.012 0.000

BIO14 -0.287 -0.179 -0.109 -0.014 -0.172 -0.234 0.325 0.213 0.152 -0.028 0.168 -0.284 0.165 -0.176 -0.572 0.140 0.308 -0.078 0.000

BIO15 0.151 0.189 0.104 -0.570 0.191 0.430 0.572 0.216 0.042 -0.033 -0.087 0.009 0.003 0.016 0.010 -0.012 0.007 0.003 0.000

BIO16 -0.276 -0.133 -0.156 -0.269 0.089 0.175 -0.184 -0.225 -0.150 0.124 -0.034 0.029 0.408 0.426 -0.259 0.334 -0.346 0.000 0.000

BIO17 -0.288 -0.177 -0.112 -0.008 -0.176 -0.213 0.282 0.211 0.157 -0.025 0.069 -0.295 0.098 0.041 0.588 0.032 -0.439 0.065 0.000

BIO18 -0.253 -0.096 -0.210 -0.326 0.292 -0.203 -0.229 0.013 0.420 -0.505 -0.205 0.240 -0.250 -0.055 -0.016 0.007 0.004 -0.024 0.000

BIO19 -0.292 -0.177 -0.062 0.006 -0.253 0.027 0.205 0.128 -0.322 0.212 -0.020 0.562 -0.467 -0.131 -0.075 0.158 -0.157 -0.031 0.000

% 50.35 22.29 14.69 8.00 1.88 1.08 0.84 0.39 0.28 0.12 0.04 0.02 0.02 0.01 0.00 0.00 0.00 0.00 0.00 100.0 Variance

210 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

Table S4 Loadings matrix obtained by the principal components analysis corrected by phylogeny of 19 bioclimatic variables on Carex maritima, C. incurviformis, C. pseudofoetida and C. sajanensis using the reduced data set.

PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PC11 PC12 PC13 PC14 PC15 PC16 PC17 PC18 PC1 9 BIO1 0.729 -0.191 -0.648 -0.019 -0.072 0.008 -0.050 0.035 0.010 0.000 -0.021 0.043 -0.002 0.010 0.015 -0.001 -0.001 0.001 0.000

BIO2 -0.481 0.135 -0.476 0.179 0.557 -0.274 -0.281 -0.076 0.062 0.049 0.073 0.015 0.095 0.009 -0.005 -0.024 -0.013 0.006 0.000

BIO3 0.789 0.053 -0.188 0.118 0.326 -0.128 -0.228 -0.190 0.019 0.132 0.095 0.003 0.234 0.050 0.048 0.160 0.024 -0.047 0.000

BIO4 -1.000 -0.008 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

BIO5 -0.211 -0.241 -0.934 0.007 -0.020 -0.032 -0.130 0.049 0.023 0.018 0.010 -0.048 -0.015 0.007 0.004 0.001 0.000 0.001 0.000

BIO6 0.943 -0.124 -0.274 -0.044 -0.124 0.047 0.011 0.016 -0.023 0.005 0.009 -0.016 0.004 0.005 0.002 -0.001 -0.001 0.001 0.000

BIO7 -0.980 0.017 -0.129 0.044 0.108 -0.058 -0.065 0.006 0.031 0.003 -0.005 -0.005 -0.010 -0.002 0.000 0.001 0.001 0.000 0.000

BIO8 -0.331 0.001 -0.840 -0.347 0.015 -0.068 0.112 -0.213 0.032 -0.012 0.002 0.001 -0.006 0.002 -0.002 0.000 0.000 -0.001 0.000

BIO9 0.875 -0.072 -0.344 0.313 0.073 0.001 0.080 0.000 -0.013 -0.009 -0.002 -0.002 0.001 0.001 0.000 0.000 0.000 0.000 0.000

BIO10 0.015 -0.299 -0.938 -0.025 -0.143 0.016 -0.067 0.058 -0.020 0.002 0.002 0.021 -0.002 -0.025 -0.015 0.002 -0.012 -0.010 0.000

BIO11 0.925 -0.114 -0.356 -0.017 -0.062 0.010 -0.027 0.015 -0.006 -0.001 0.001 0.006 0.008 -0.007 -0.006 0.000 0.007 0.003 0.000

BIO12 0.791 -0.611 0.023 0.002 -0.004 0.001 -0.002 -0.002 0.005 -0.003 -0.003 -0.002 0.001 0.000 0.000 0.000 0.000 0.000 0.000

BIO13 0.751 -0.606 -0.083 -0.056 0.147 0.177 0.014 -0.036 -0.015 0.004 0.047 -0.004 -0.004 -0.034 0.027 0.000 0.000 0.002 0.000

BIO14 0.754 -0.625 0.088 0.016 -0.065 -0.159 -0.013 0.004 -0.023 -0.031 0.028 0.013 -0.013 -0.002 -0.009 0.020 -0.014 0.021 0.000

BIO15 -0.605 0.250 -0.060 -0.093 0.403 0.111 -0.429 -0.207 -0.381 -0.088 -0.067 -0.016 -0.002 0.004 0.001 0.000 0.000 0.000 0.000

BIO16 0.763 -0.618 -0.051 -0.046 0.109 0.133 -0.007 0.011 0.004 -0.004 0.018 0.009 -0.007 0.007 -0.005 0.001 0.001 0.000 0.000

BIO17 0.762 -0.619 0.078 0.022 -0.060 -0.151 -0.006 0.012 -0.024 -0.028 0.028 0.007 -0.006 0.002 0.002 -0.001 0.002 -0.002 0.000

BIO18 0.685 -0.649 -0.125 -0.225 0.150 -0.088 0.084 0.063 -0.041 0.030 -0.006 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

BIO19 0.810 -0.555 0.098 0.129 -0.061 -0.009 -0.021 -0.046 -0.018 0.043 0.004 0.006 -0.007 0.001 -0.001 0.000 0.000 0.000 0.000

% 50.35 22.29 14.69 8.00 1.88 1.08 0.84 0.39 0.28 0.12 0.04 0.02 0.02 0.01 0.00 0.00 0.00 0.00 0.0 10 Varia 0 0.0 nce

211 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

Table S5 Loadings matrix obtained by the principal components analysis of 19 bioclimatic variables on Carex maritima full data set.

PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PC11 PC12 PC13 PC14 PC15 PC16 PC17 PC18 PC19 -0.258 0.166 -0.207 -0.090 0.004 0.121 0.014 0.083 -0.037 -0.115 0.215 0.277 -0.199 -0.052 0.559 0.535 0.223 0.074 0.000 BIO1 0.151 0.341 -0.046 0.620 0.160 -0.185 0.379 -0.027 -0.056 -0.351 0.278 0.125 -0.065 0.002 -0.212 0.037 -0.017 0.003 0.000 BIO2 -0.174 0.032 -0.315 0.657 -0.274 0.059 -0.399 0.025 0.165 0.367 -0.187 0.011 -0.006 -0.004 0.050 -0.008 0.004 0.003 0.000 BIO3 0.255 0.127 0.265 -0.014 0.160 -0.012 -0.171 -0.113 0.041 0.279 -0.108 0.153 -0.115 0.025 -0.307 0.478 0.258 -0.512 0.000 BIO4 -0.080 0.554 -0.026 -0.031 0.336 0.118 -0.141 0.131 0.012 -0.075 -0.186 -0.480 0.406 0.129 0.104 0.049 0.100 0.010 0.216 BIO5 -0.268 -0.001 -0.220 -0.099 -0.083 0.107 -0.013 0.133 -0.002 -0.173 -0.011 -0.151 0.105 0.098 -0.444 0.231 0.100 0.078 -0.703 BIO6 0.252 0.178 0.220 0.093 0.193 -0.073 -0.032 -0.096 0.006 0.155 -0.048 0.004 0.020 -0.061 0.494 -0.224 -0.072 -0.078 -0.677 BIO7 0.023 0.498 -0.007 -0.249 -0.640 -0.425 0.016 -0.290 0.100 -0.035 -0.055 -0.013 0.011 0.002 -0.007 -0.003 -0.002 0.001 0.000 BIO8 -0.260 0.030 -0.220 -0.008 0.139 0.140 0.559 -0.507 -0.218 0.386 -0.270 -0.002 0.055 -0.018 -0.015 0.024 -0.004 -0.004 0.000 BIO9 -0.159 0.458 -0.054 -0.211 0.249 0.217 -0.251 0.014 -0.001 0.162 0.106 0.355 -0.349 -0.102 -0.277 -0.296 -0.224 0.187 0.000 BIO10 -0.266 0.032 -0.236 -0.059 -0.072 0.099 0.044 0.091 -0.033 -0.180 0.138 -0.011 -0.012 -0.067 0.081 -0.335 -0.035 -0.817 0.000 BIO11 -0.264 -0.012 0.263 0.053 0.007 -0.017 -0.014 -0.022 0.122 -0.061 -0.040 0.150 0.290 -0.047 0.023 0.345 -0.773 -0.107 0.000 BIO12 -0.251 0.027 0.324 0.062 -0.046 0.088 0.201 0.010 0.396 0.202 0.303 -0.541 -0.410 -0.155 -0.005 0.048 -0.005 0.008 0.000 BIO13 -0.264 -0.063 0.206 0.082 0.016 -0.142 -0.304 -0.298 -0.501 0.070 0.407 -0.128 -0.023 0.485 0.019 -0.028 0.004 -0.001 0.000 BIO14 0.202 0.143 0.296 0.113 -0.438 0.732 0.063 -0.021 -0.295 -0.129 -0.044 -0.010 0.009 -0.012 0.009 -0.001 -0.017 0.010 0.000 BIO15 -0.254 0.034 0.314 0.020 -0.068 0.071 0.186 0.124 0.331 0.192 0.209 0.401 0.465 0.197 -0.029 -0.206 0.352 0.048 0.000 BIO16 -0.265 -0.048 0.228 0.088 0.047 -0.111 -0.219 -0.197 -0.230 -0.153 -0.002 0.000 0.193 -0.760 -0.073 -0.037 0.255 0.061 0.000 BIO17 -0.249 0.091 0.272 0.060 -0.088 -0.277 0.202 0.592 -0.403 0.143 -0.374 0.040 -0.221 0.039 0.009 -0.029 0.022 -0.014 0.000 BIO18 -0.262 -0.068 0.230 0.094 0.106 0.046 -0.090 -0.302 0.270 -0.485 -0.491 0.103 -0.294 0.268 0.058 -0.106 0.138 0.004 0.000 BIO19 % of 65.9 14.7 9.4 4.7 2.3 1.7 0.4 0.3 0.2 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 100. Varia 0 nce

212 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

Table S6 Bioclimatic variables used. Units of bioclimatic variables are °C × 10 for temperature (excluding BIO4 which was calculated based on K × 10 to deal with negative temperatures) and mm for precipitation.

Bioclimatic variable Description BIO1 annual mean temperature BIO2 mean diurnal temperature range [mean of monthly (maximum temperature - minimum temperature)] BIO3 isothermality (BIO2 / BIO7 x 100) BIO4 temperature seasonality (standard deviation of monthly temperature) BIO5 maximum temperature of the coldest month; BIO6 minimum temperature of the warmest month BIO7 temperature range (BIO6 - BIO5) BIO8 mean temperature of the wettest quarter BIO9 mean temperature of the driest quarter BIO10 mean temperature of the warmest quarter BIO11 mean temperature of the coldest quarter BIO12 annual precipitation BIO13 precipitation of the wettest month BIO14 precipitation of the driest month BIO15 precipitation seasonality (coefficient of variation of monthly precipitation) BIO16 precipitation of the wettest quarter BIO17 precipitation of the driest quarter BIO18 precipitation of the warmest quarter BIO19 precipitation of the coldest quarter

213 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

Table S7 Area under the curve (AUC) ± standard deviation (SD) and the variables that contributed the most to explain MAXENT models under WorldClim database layers and the most important when used alone, for each group of Carex maritima.

AUC ± SD Environmental The most important variables that environmental contributed most variable when used alone

C. maritima from both 0.941 ± 0.002 Annual mean BIO1 hemispheres temperature (BIO1)

C. maritima from Northern 0.942 ± 0.002 BIO1 BIO1 Hemisphere C. maritima from Southern 0.997 ± 0.003 minimum temperature BIO6 Hemisphere of the warmest month (BIO6)

214 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

REFERENCES

Blattner F.R. (1999) Direct amplification of the entire ITS region from poorly preserved plant material using recombinant PCR. Biotechniques, 1180–1186.

Brummitt R.K. (2001) World geographical scheme for recording plant distributions, 2nd edn. Hunt Institute for Botanical Documentation, Pittsburgh, PA.

Escudero M. & Luceño M. (2009) Systematics and evolution of Carex sects. Spirostachyae and Elatae (Cyperaceae). Plant Systematics and Evolution, 279, 163–189.

Shaw J., Lickey E.B., Beck J.T., Farmer S.B., Liu W., Miller J., Siripun K.C., Winder C.T., Schilling E.E. & Small R.L. (2005) The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. American Journal of Botany, 92, 142–166.

Starr J.R., Harris S.A., & Simpson D.A. (2003) Potential of the 5 and 3 ends of intergenic spacer (IGS) of rDNA in the Cyperaceae: new sequences for lower-level phylogenies in sedges with an example from Uncinia Pers. International Journal of Plant Sciences, 164, 213–227.

White T.J., Bruns T., Lee S., & Taylor J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications (ed. by M. Innis, D. Gelfand, D. Sninsky and T. White), pp. 315–322. Academic Press, San Diego, CA.

215 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

216 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

Journal of Biogeography

SUPPORTING INFORMATION

Long-distance dispersal during the middle–late Pleistocene explains the bipolar disjunction of Carex maritima (Cyperaceae)

Tamara Villaverde, Marcial Escudero, Modesto Luceño and Santiago Martín-Bravo

Appendix S2 Supplementary figures. Distribution map of Carex maritima (Fig. S1); ecological niche models of C. maritima groups

fitted to current climatic conditions from MAXENT analyses (Fig. S2), and projections of the models to Last Glacial Maximum (18–21 ka; MIROC and CCSM models) and Last Interglacial Period (c. 120–140 ka) (Fig. S3); scatter plots of the uncorrelated

bioclimatic variables used in MAXENT analyses (Fig. S4); and scatter plot of the three first components from the principal components analysis of the full data set (Fig. S5).

217 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

Figure S1 Distribution map of Carex maritima (894 preserved specimens) depicted by black circles, obtained from occurrence data between 1950 and 2000, downloaded from the Global Biodiversity Information Facility data portal (http://www.gbif.org/, downloaded 22 December 2014) after pruning for likely incorrect identification or georeferencing (e.g. occurrences in oceans) and removing duplicate records from the same locality to reduce the effects of spatial autocorrelation.

218 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

(a)

(b)

219 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

(c)

Figure S2 Ecological niche model of Carex maritima groups fitted to current climatic conditions (c. 1950–2000) from MAXENT analyses. Colours correspond to a continuous prediction with values ranging from 0 to 1 (from white to red, respectively). (a) Carex maritima; (b) C. maritima populations of the Northern Hemisphere; (c) C. maritima populations of the Southern Hemisphere.

220 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

Figure S3 Maps of predicted environmental suitability for Carex maritima and population groups (Northern and Southern

Hemispheres). MAXENT projections of the model to the Last Glacial Maximum (LGM, 18–21 ka; MIROC and CSSM model shown) and Last Interglacial Period (LIG, c. 120–140 ka). Colours correspond to a continuous prediction with values ranging from 0 to 100 (from blue to red, respectively).

221 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

(a)

222 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

(b)

223 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

(c)

Figure S4 Scatter plots of the uncorrelated bioclimatic variables used in MAXENT analyses of Carex maritima Northern and Southern Hemisphere samples (grey and red circles, respectively): (a) annual mean temperature (BIO1) and minimum temperature of the warmest month (BIO6); (b) BIO1 and annual precipitation (BIO12); and (c) BIO6 and BIO12.

224 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

(a)

225 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

(b)

226 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

(c)

Figure S5 Scatter plots of the first three components explaining up to 90% of the observed variance (see Appendix S1), derived from the principal components analysis of the full data set (894 populations) and depicting in a climate-niche space the position of Carex maritima Northern and Southern Hemisphere samples (grey and red circles, respectively): (a) principal component 1 (PC1) and principal component 2 (PC2); (b) PC1 and principal component 3 (PC3); and (c) PC2 and PC3.

227 Chapter 4. Long-distance dispersal during the middle–late Pleistocene explains the bipolar ______disjunction______of Carex maritima

228 Chapter 5

Two independent dispersals to the Southern Hemisphere to become the most widespread Carex bipolar species: biogeography of C. canescens (Cyperaceae)

229 230 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

Two independent dispersals to the Southern Hemisphere to become the most

widespread Carex bipolar species: biogeography of C. canescens (Cyperaceae)

Tamara Villaverdea, Marcial Escuderob,c, Santiago Martín-Bravoa and Modesto Luceñoa

aDepartment of Molecular Biology and Biochemical Engineering, Pablo de Olavide

University, Seville, Spain. bDepartment of Integrative Ecology, Estación Biológica de

Doñana (EBD-CSIC), Seville, Spain. cDepartment of Plant Biology and Ecology,

University of Seville, Seville, Spain.

*Correspondence: Tamara Villaverde. Department of Molecular Biology and

Biochemical Engineering, Pablo de Olavide University, carretera de Utrera Km 1 sn

41013 Seville, Spain. email: [email protected]

231 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

Abstract

Aim: To test the various hypotheses accounting for the bipolar disjunction of Carex

canescens and to elucidate if it migrated twice to the Southern Hemisphere or if it

dispersed from South America to Australia (or vice versa).

Location: Arctic/boreal latitudes of both hemispheres.

Methods: We obtained and analysed DNA sequences for the nuclear internal and

external transcribed spacers (ITS and ETS) and for the plastid 5′ trnK and rps16 introns

from 56 populations of C. canescens and 8 populations from its sister species and

outgroups. We also climatically characterized the species distribution by adding 1,995

species presence data points from the Global Biodiversity Information Facility and

using the climatic information stored in the WorldClim database.

Results: Although the internal phylogenetic resolution of C. canescens was poor and

populations were embedded in a polytomy independently of their geographical origin,

genetic structure was detected between South America and Australia, which did not

share any of the sampled haplotypes. The diversification of C. canescens occurred

during the Pleistocene (1.17 Ma; 95% HPD 0.34 – 2.17 Ma). Southern Hemisphere

populations occupy a more restricted climatic niche than in the Northern Hemisphere

but falling within the general ecological conditions tolerated by the species, which seem

to be very wide.

Main conclusions: Carex canescens dispersed twice from the Northern Hemisphere to

South America and Australia. Recent divergence times and the lack of genetic

differentiation between disjunct populations did not allow us to discern between direct

dispersal and mountain-hopping or a combination of both, to explain the colonization of

the Southern Hemisphere. Long-distance dispersal is claimed as a widespread

232 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

phenomenon in bipolar Carex species, possibly facilitated by rare processes or unusual

behaviour of vectors.

Keywords: Biogeography, climatic niche, Cyperaceae, divergence time estimation,

Glareosae, long-distance dispersal.

233 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

INTRODUCTION

Interest in establishing the relations between floras in the Southern Hemisphere has

been fostered by their similarities with floras of the Northern Hemisphere (e.g., Raven,

1963; Raven, 1972; Wen & Ickert-Bond, 2009; Leslie et al., 2012). Around 75-85% of

families present in Tasmania and South America (south of ca. 40º S) also occur in the

Northern Hemisphere, whereas ca. 20-27% of the genera are shared between New

Zealand-South America and the Northern Hemisphere (Moore, 1972). Plant disjunctions

between hemispheres are formidable when species reach very high latitudes at both

sides of the Equator, regardless of its occurrence at intermediate latitudes, thus

achieving the so-called bipolar distribution (Moore & Chater, 1971). About 30 vascular

plant species are known to have such a distribution, which are mainly restricted to

alpine and polar regions (Moore & Chater, 1971).Bipolar disjunctions have historically

been explained by four hypotheses: (1) vicariance (Du Rietz, 1940), which implies a

continuous distribution fragmentation during the Mesozoic Era (Scotese et al., 1988);

(2) convergent or parallel evolution of the disjunct populations (Hofsten, 1916;

Scotland, 2011); (3) stepwise long-distance dispersal across the Equator via mountain

ranges (“mountain-hopping”; Raven, 1963; Moore & Chater, 1971; Ball, 1990; Vollan

et al., 2006); and (4) direct long-distance seed dispersal by birds, wind and/or ocean

currents (Cruden, 1966; Muñoz et al., 2004; Nathan et al., 2008; Gillespie et al., 2012).

The genus Carex L., which consists of c. 2000 species (Reznicek, 1990), has the

greatest number of bipolar taxa (6), which generally have a circumboreal distribution

and are limited to austral latitudes in South America (>52º). An exception is C.

canescens (sect. Glareosae G. Don), the single bipolar Carex species that reaches not

only the southernmost region of South America (Tierra del Fuego and Falkland Islands)

but also Oceania (including Australia, Tasmania and New Guinea; Fig. 1 and Appendix

234 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

S1), occurring within five biogeographical regions (Nearctic, Palearctic, Andean,

Neoguinean and Australotemperate; Morrone, 2002). Carex canescens is therefore the

bipolar Carex species with the widest distribution.

The great morphological variability within C. canescens across its wide

distribution has motivated the description of more than 40 infraspecific taxa, of which

only Carex canescens subsp. disjuncta (Fernald) Toivonen (that occurs in eastern North

America) and C. canescens var. robustior Blytt ex Andersson (that is distributed in

Patagonia and Falkland Islands) are currently accepted (Govaerts et al. 2014). Carex

canescens var. robustior is also considered an ecotype of C. canescens from the

mountainous regions in North America, but this taxon is not currently accepted in North

America (Toivonen, 2002). A morphological study of C. canescens covering its total

range found slight differences between Northern and Southern Hemisphere populations,

which only deserved varietal recognition (Moore & Chater, 1971). In general, Southern

Hemisphere plants tend to be greater than their Northern Hemisphere counterparts for

various parts (e.g., stem diameter, leaf size and utricle width), although there is some

overlapping (Moore & Chater, 1971). Moore (1972) interpreted that these

morphological differences between hemispheres could suggest a transtropical migration

in the Americas and subsequent circum-Antarctic dispersal. Nelmes (1951) proposed

that C. canescens populations from Malaysia are intermediate forms between Australian

and European forms based on the number and conspicuously (or not conspicuously)

nerved utricles, and suggested a southward migration and adaptation of southern

populations (Moore, 1972). In C. canescens, as well as in other temperate sedges, leaf

elongation has been proven to increase with temperature (Heide, 1997, 2000) and might

not have a genetic origin. Vollan et al. (2006) analysed samples of C. canescens from

Europe (only Norway), South America (Chile) and Australia using amplified fragment

235 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

length polymorphisms (AFLPs) and found genetic differentiation between Northern and

Southern Hemisphere populations. They hypothesized a Northern Hemisphere origin

and a single colonization of the Southern Hemisphere, followed by secondary dispersal

from Australia to South America or vice versa. Escudero et al. (2010a) found that the

genetic distance between populations of C. canescens from North and South America

was lower than between some populations from the Northern Hemisphere, which could

indicate a more recent connection between North America and Patagonia than among

some areas of the Northern Hemisphere. However, sampling was limited in both of

these studies, including only six or seven populations and lacking samples from North

America (Vollan et al., 2006) or Australia (Escudero et al., 2010a). For bipolar Carex

species, the vicariance hypothesis (Du Rietz, 1940) is rejected as the origin of the family

Cyperaceae (82.6 Ma, 95% highest posterior density: 75.9–85.6 Ma; Escudero & Hipp,

2013) is placed during the Cretaceous, which is well after the fragmentation of the

trans-tropical highland bridges that occurred during the Mesozoic Era (195 Ma; Scotese

et al., 1988).

Here we aimed to: (i) test the various hypotheses accounting for the bipolar

disjunction of C. canescens; and (ii) to determine whether C. canescens migrated twice

to the Southern Hemisphere or was dispersed from South America to Australia or vice

versa.

MATERIALS AND METHODS

Study species and sampling

Carex canescens is distributed in the temperate areas of both hemispheres, with a

circumpolar range in the Northern Hemisphere, whereas its range is limited to Patagonia

and south-eastern Australia in the Southern Hemisphere (Fig. 1). Potential C. canescens

236 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

herbarium specimens from New Guinea were depauperate and infertile, precluding a

confident identification. Carex canescens is a wind-pollinated herbaceous

hemicryptophyte that usually grows in persistently wet, base-poor sites (e.g., sphagnum

bogs), moist coniferous forests, woodlands, meadows, lakeshores, rivers and other fresh

water bodies. We obtained plant material representing the entire range of C. canescens

subsp. canescens (58 populations) as circumscribed by Egorova (1999) and Toivonen

(2002). We also included one population each of C. canescens subsp. disjuncta

(Fernald) Toivonen, C. lachenalii Schkuhr, C. glareosa Schkuhr ex Wahlenb., C. furva

Webb and C. arcta Boott from section Glareosae. We use C. macloviana d’Urv., C.

maritima Gunn., C. paniculata subsp. paniculata L. and C. remota L. as outgroups

(Waterway et al. 2009). For all species one individual per population was sampled.

Samples used for the molecular study were obtained from silica-dried leaf material

collected in the field and from herbarium specimens (Appendix S1). Vouchers for new

collections are deposited in the following herbaria: CAN, SI and UPOS (abbreviation

following Index Herbariorum).

PCR amplification and sequencing

Total DNA was extracted using DNeasy Plant Mini Kit (Qiagen, California). Forward

and reverse primers were used for amplifications of the internal transcribed spacer

region (ITS: ITS-A, ITS-4; White et al., 1990; Blattner, 1999), external transcribed

spacer regions (ETS: ETS-1f, 18S-R; Starr et al., 2003), 5′trnK intron (5′trnKCarexF,

5′trnKCarexR; Escudero & Luceño, 2009) and rps16 intron (rps16F-rps16R; Shaw et

al., 2005). Amplifications were obtained in a Perkin Elmer PCR-system 9700

(California) under the conditions specified by Escudero et al. (2010a) for ITS and

rps16, Starr et al. (2003) for ETS, and Escudero & Luceño (2009) for 5′trnK intron.

237 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

Polymerase chain reaction (PCR) products were cleaned and sequenced following

Escudero et al. (2008). Sequences were edited, automatically aligned with MUSCLE

(Edgar, 2004) and manually adjusted using GENEIOUS v6.1.7 (Biomatters, Auckland,

New Zealand). We coded three informative indels for the 5′trnK region following the

simple gap coding method by Simmons & Ochoterena (2000).

Phylogenetic analyses

We obtained a total of 56 sequences of ITS (six from NCBI GenBank), 41 of ETS (four

from NCBI GenBank), 43 of rps16 (two from NCBI GenBank) and 47 of 5′trnK (one

from NCBI GenBank; Appendix S1). Each of the four loci was analysed independently

and in combination using Maximum Likelihood (ML) and Bayesian inference (BI). The

combined nuclear and plastid matrix consisted of 64 combined sequences with 2,635

sites (see Appendix S1). Maximum likelihood and Bayesian analyses were performed as

described by Villaverde et al. (2015a). Selected nucleotide substitution models under

the Akaike Information Criterion (AIC) in jModeltest (Posada, 2008) were GTR+G for

ITS1 and ITS2, HKY for ITS 5.8s; GTR for rps16 and HKY+I for 5′trnK (Appendix

S1).

Haplotype network and divergence time estimation

We obtained the genealogical relationships among ptDNA haplotypes using the plastid

combined matrix and statistical parsimony as implemented in TCS v1.21 (Clement et

al., 2000) and described in Villaverde et al. (2015a). We estimated completeness of

haplotype (5′trnK- rps16) sampling using a Stirling probability distribution, as

described by Dixon (2006), which calculates a posterior probability distribution of the

total number of haplotypes (sampled or not).

238 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

Dated phylogenies were estimated for the nuclear and plastid matrix in BEAST

v1.7.5 (Drummond et al., 2012). All phylogenies were estimated using an uncorrelated

log-normal relaxed clock model. A normal age prior with a mean of 14.82 million years

ago (Ma) ± 2.5 million years (Myr) was applied to the root of the tree based on previous

estimates (Escudero & Hipp, 2013). Analyses were conducted using two independent

MCMC runs of 40 million generations each, assuming the Birth Death tree prior with a

mean substitution rate set at 1.0. Run convergence and burn-in were assessed in TRACER

v1.5 (Rambaut & Drummond, 2009). Maximum Clade Credibility (MCC) trees were

calculated with TREEANNOTATOR v1.7.2 (Drummond & Rambaut, 2007) using a

posterior probability limit of 0.7 and the mean heights option.

Climatic environment

We obtained values for 19 bioclimatic variables (Appendix S1) as described by

Escudero et al. (2013) for each sampled population of C. canescens, except for one

population from the USA (Wisconsin) lacking precise geographic coordinates. This data

set was completed by adding: (i) five additional populations from Australia for which

we failed to amplify any loci (see Appendix S1); (ii) 1,992 species occurrence records

originated from herbarium specimens, collected between 1950 and 2014, and

downloaded from the Global Biodiversity Information Facility data portal

(http://www.gbif.org/, downloaded 12 February 2015). This dataset was refined by

removing likely incorrectly identified (e.g., occurrences outside of the distribution range

defined by Monocot checklist, Govaerts et al. 2014) or incorrectly georeferenced

populations (e.g., occurrences in oceans) and duplicate records from the same locality to

reduce the effects of spatial autocorrelation. Our final data set included a total of 2,057

populations (Appendix S2). To characterize its climatic niche space, we performed a

239 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

principal components analysis (PCA) of the climatic dataset using the prcomp function

(sdev, rotation, centre and scale options were set as TRUE) in R (R Core Team, 2014).

Ecological niche model

A species distribution modeling was performed to reconstruct the potential ranges of C.

canescens under present clime with MAXENT v3.3.3k (Phillips et al., 2006). We

performed a correlation analysis with the Variance Inflation Factor (VIF) using the vif

function in the usdm package in R (R Development core Team, 2014) and a correlation

threshold of 0.7 (Dormann et al., 2013) . Only three variables were uncorrelated and

consequently included in the analyses: bio1 (annual mean temperature), bio6 (minimum

temperature of the warmest month) and bio12 (annual precipitation). Replicate runs

(500) were performed using the bootstrap run type. Analyses were performed for all

populations of C. canescens and separately by Northern and Southern Hemisphere

populations, as well as by populations of C. canescens from South America and

Australia. We partitioned all the locality data into training and testing data sets (75% vs.

25%, respectively) in order to build niche models and to evaluate the quality of the

model.

RESULTS

Phylogenetic reconstruction

Carex section Glareosae was obtained as monophyletic in all the analyses with a strong

support (94% BS / 1 PP, Appendix S2). The monophyly of C. canescens was strongly

supported (97% BS / 1PP), with the single sampled population of C. canescens subsp.

disjuncta retrieved as sister to the remainder of C. canescens subsp. canescens. The

240 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

internal phylogenetic resolution of the species was poor and populations from different

hemispheres or continents did not form clades. Most populations were embedded in a

polytomy, independently of their geographical origin, with the exception of two samples

from South America, which formed a weakly to moderately supported clade (61% BS /

0.84 PP, Appendix S2).

Haplotype network

The obtained ptDNA (5′trnK-rps16) haplotype network revealed five different

haplotypes (H1-H5) and one missing haplotype (Fig. 3). A probability of 95% that all

haplotypes have been sampled is given by Dixon’s (2006) method. Haplotype 1 (H1) is

shared by 34 samples from Australia, Eurasia and North America, while haplotype 2

(H2), separated by one mutation from H1, is shared by 11 samples from Eurasia, North

and South America. There are three haplotypes exclusive to single populations: one

from western North America (California; H3), one from South America (Argentina,

Santa Cruz; H4) and one from C. canescens subsp. disjuncta from north-eastern North

America (Massachusetts; H5). No more than one or two mutations are needed to

connect these singletons with H1 or H2 (Fig. 3). A loop connects H1, H2, H4 and the

missing haplotype that leads to H5, which reflects ambiguity about the evolutionary

history of these haplotypes due to homoplasy. Four of the five haplotypes (H1-H3, H5)

were found in North America, whereas two in Eurasia (H1, H2) and South America

(H2, H4) and only one in Australia (H1). Interestingly, populations from the Northern

Hemisphere shared haplotypes with those from the Southern Hemisphere (H1, H2),

whereas different haplotypes were found in Australia (H1) versus South America (H2,

H4).

241 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

Divergence time estimation

The dating analyses produced a congruent topology with respect to the BI and ML

analyses presented above (Fig. 2 and Table 1). The divergence time of the clade

comprising section Glareosae was 6.55 Ma (95% highest posterior density, HPD, 2.56 –

10.99 Ma), which falls in the Late Miocene - Pliocene. Thus, the diversification of C.

canescens could have occurred during the Pleistocene (1.17 Ma; 95% HPD 0.34 – 2.17

Ma). The clade comprised by C. lachenalii and C. glareosa diverged during the same

Epoch (1.03 Ma, 95% HPD 0.18 – 2.14 Ma).

Climatic environment

The PCA of the climatic data set showed that PC1 explained 48.1% of the variance,

whereas PC2 and PC3 explained 15.7 and 14.5% respectively (Appendix S1). The

variables with the highest loadings in PC1 were annual precipitation (bio12),

temperature seasonality (bio4) and precipitation of coldest quarter (bio19; Appendix

S1). The scatter plot of the three first components (Fig. 4) is coloured by geographic

groups corresponding to: C. canescens samples from North-East (Eurasia), North-West

(North America), South-East (Australasia) and South-West (South America) quadrants.

Separation between the Northern and Southern Hemisphere samples of C. canescens or

between different landmasses within the same hemisphere was not observed (Fig. 4).

Present distribution under climatic change scenarios

Current conditions

Our results show that the modelled ecological niche of C. canescens, including the

Northern and Southern Hemisphere samples, predicts suitable areas in both

hemispheres. The environmental variable that contributed most to explain the

242 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

MAXENT models under WorldClim database layers and the most important

environmental variable when used alone, according to the jack-knife test, was annual

mean temperature (bio1). The modelled ecological niche of C. canescens including only

the Northern Hemisphere populations predicts suitable habitats also in the Southern

Hemisphere (Appendix S2). Values for the area under the curve (AUC) were all above

0.89, which indicate a good fit of the models. The average AUC for each group and the

most important environmental variables detected in each analysis are reported in

Appendix S1.

DISCUSSION

Our results show that C. canescens is a monophyletic species, which allows us to

strongly reject the convergent or parallel evolution hypotheses for the origin of the

bipolar disjunction (Hofsten, 1916; Scotland, 2011), since we would expect two or more

lineages of C. canescens with different common ancestors under these hypotheses.

Carex canescens diverged from its sister species in section Glareosae 1.17 Ma (95%

HPD: 0.34 – 2.17) and the clade formed by only two samples from South America is of

recent origin (Fig. 3). In addition, no clear genetic differentiation was found between the

other Northern and Southern Hemisphere populations, since they shared haplotypes

(H1, H2; Fig. 2), which also points to a rather recent origin of the bipolar disjunction.

Therefore, the remaining alternative hypotheses are mountain-hopping through the

American Cordillera and Malaysian mountains, direct long-distance dispersal, or a

combination of both.

Based on our haplotype network (Fig. 2), we cannot infer the direction of

dispersal (North-to-South or South-to-North). The haplotype with the highest number of

mutational connections (three), which is considered the ancestral haplotype under the

243 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

coalescent theory (Posada & Crandall 2001), comprises populations from Eurasia,

North and South America (H2). Nevertheless, the haplotype H1 could also be ancestral,

as it is also widely distributed (Eurasia, North America and Australia) and has only one

connection less than H2. All 23-25 species of Carex section Glareosae (Maguilla et al.,

in press), with the exception of C. canescens and C. lachenalii, are exclusively

distributed in the Northern Hemisphere, which supports the hypothesis of a northern

origin of the species and a subsequent North-to-South direction of dispersal. The same

hypothesis is supported by the widespread distribution of C. canescens in the Northern

Hemisphere relative to its very restricted distribution in the Southern Hemisphere (Fig.

1). Other works on bipolar taxa (including two examples of bipolar Carex species) have

also shown a predominantly southwards colonization route (Gussarova et al., 2008;

Popp et al. 2011; Piñeiro et al., 2012; Villaverde et al., 2015a; 2015b). Therefore, we

suggest that C. canescens’ distribution is a result of long-distance dispersal from the

Northern Hemisphere to the Southern Hemisphere.

Not once but twice: the double colonization of the Southern Hemisphere by C.

canescens

While haplotype sharing was found between Northern and Southern Hemisphere

populations, genetic structure was detected between South America and Australia,

which did not share any of the sampled haplotypes (Fig. 2). Therefore, we can infer at

least two different colonization events of C. canescens from the Northern to the

Southern Hemisphere, one to each of these two southern landmasses. When taxa known

to have originated from the Northern Hemisphere are distributed in two landmasses

within the Southern Hemisphere, it can be either the result of a single colonization of

the Southern Hemisphere followed by subsequent dispersal to the other landmass (e.g.,

244 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

Schuettpelz & Hoot, 2004; Inda et al., 2008; Gussarova et al., 2008; Nie et al., 2012) or

of multiple independent dispersals from the Northern Hemisphere (e.g., Yokoyama et

al., 2000; Escudero et al., 2009; Schaefer et al., 2009; Emadzade et al. 2011; Banasiak

et al., 2013).

Two different mountain ranges connect the Northern and Southern Hemispheres,

allowing migration of cold-adapted plant species by mountain-hopping. The mountain

uplift in Malaysia in the Miocene-Pliocene (10 Ma; Sanmartín & Ronquist, 2004) and

the gradually cooler climate in both hemispheres at that time (Scotese, 2002) could have

facilitated the dispersal of cold-adapted species into the Southern Hemisphere from

Eurasia (Smith, 1981). Similarly, the American cordillera has connected North and

South America since the Late Miocene (Smith, 1986) and it has acted as a corridor for

the dispersal of different organisms (e.g., Moreno et al., 1994; Antonelli & Sanmartín,

2011). The absence of genetic differentiation between the Southern and the Northern

Hemisphere and its diversification time suggest a very recent dispersal of C. canescens

to the Southern Hemisphere. The same pattern of genetic similarity between

Hemispheres was found in another bipolar Carex species (C. arctogena; Villaverde et

al., 2015b), for which direct long-distance dispersal best explains the bipolar

disjunction. A different genetic pattern was, however, found for C. maritima (Villaverde

et al., 2015a), whose populations from the Southern Hemisphere are genetically distinct

from the Northern populations. The obtained results for C. maritima did not allow us to

discern between direct long-distance dispersal or mountain hopping. Likewise, we

consider that the current study is not conclusive enough to be able to distinguish

between mountain-hopping or direct long-distance dispersal to explain C. canescens’

distribution.

245 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

The high colonization capacity and competitiveness of C. canescens

None of the six bipolar species in the genus are known to occur in two landmasses

within the Southern Hemisphere, as well as the Falkland Islands, New Guinea and

Tasmania. The migration to such islands supports the high dispersal and colonization

ability of C. canescens, ranked as the bipolar Carex species with the greatest

distribution. With the exception of C. microglochin, none of the bipolar Carex species

displays standard morphological syndromes for long-distance dispersal (as described by

Higgins et al., 2003). Some dispersal agents might have been involved in the

distribution of C. canescens, such as birds, ocean or wind currents (Nathan et al., 2008;

Gillespie et al. 2012). Rare processes or unusual behaviour of vectors have been

inferred to have dispersed seeds over long distances (Higgins & Richardson, 1999;

Nathan et al., 2002; Higgins et al., 2003; Nathan et al., 2008). In fact, long-distance

dispersal is claimed as a widespread phenomenon in many plant species without

standard morphological syndromes for long-distance dispersal (Carlquist, 1967; Cain et

al., 2000; Higgins et al., 2003; Alsos et al., 2007; Dixon et al., 2009).

Establishment of plants following long-distance dispersal is determined by the

environment and biotic conditions of the host community, as well as by the colonization

capacity and competitiveness of the new hosted species. Our results show that in the

Southern Hemisphere C. canescens occupies a more restricted climatic niche than in the

Northern Hemisphere (Fig. 4 and Appendix S2), but falling within the general

ecological conditions tolerated by the species, which seem to be very wide (Fig. 4).

Furthermore, Carex species are generally highly self-pollinated (Whitkus, 1988;

Ohkawa et al., 2000; Arens et al., 2005; Friedman & Barrett, 2009; Escudero et al.,

2010b, 2013), which could also explain, at least in part, successful establishment after

dispersal (Carlquist, 1983; Baker, 1955). In addition, chromosome rearrangements have

246 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

been shown to be correlated with phenotypic differences, as well as being thought to

enhance fitness in different habitats (Coghlan et al., 2005). Specifically, high

chromosome number evolution is a result of: (i) selection by climatic regime and

ecological strategies; and (ii) neutral processes such as phylogenetic inertia or migration

processes (Escudero et al., 2012, 2013). The high chromosome number variation in C.

canescens (2n = 52-54, 56-58, 60, 62; reviewed in Roalson et al., 2008) might also be a

result of the influence of the climatic environments at different latitudes and distant

regions.

CONCLUSIONS

Our results suggest that C. canescens originated during the Pleistocene and that its

disjunction could be explained by long-distance dispersal from the Northern to the

Southern Hemisphere. Carex canescens could have dispersed at least twice to the

Southern Hemisphere, once to Australia and once to South America, either by

mountain-hopping through the Andes cordillera and Malaysian mountains, by direct

jump or by a combination of both.

ACKNOWLEDGEMENTS

The authors thank to all staff from herbaria CAN, COLO, E, M, MSB, SI, UPOS and

WIN for granting us access to their collections and for providing plant material, E.

Maguilla (Universidad Pablo de Olavide, UPO) and F. Rodríguez-Sánchez (Estación

Biológica de Doñana, EBD-CSIC) for his help with MAXENT analyses, map editing

and valuable comments on the manuscript, M. Puscas (Babes-Bolyai University) for

plant material collections, L. P. Bruederle (University of Colorado, Denver) and P.

Vargas (Real Jardín Botánico de Madrid) for assistance in plant collections, and M.

247 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

Míguez and F. J. Fernández (UPOS) for technical support. This research was supported

by the Spanish Ministry of Science and Technology through the project CGL2012-

38744 and from the Regional Ministry of Economy, Innovation, Science and

Employment of Andalusia through the project RNM-2763.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article:

Appendix S1 Studied material of Carex canesens, related species and outgroups; molecular characteristics of the amplified regions; results from the principal components analysis of 19 bioclimatic variables from the WorldClim database; description of bioclimatic variables; and AUC values from MAXENT analyses.

Appendix S2 Distribution map of Carex canescens, phylogeny of C. canescens, boxplots of the most important variables from bioclimatic principal component analyses and ecological niche models of C. canescens.

254 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

BIOSKETCH

Tamara Villaverde is a PhD student at Pablo de Olavide University, Seville (Spain). Her research is focused on the evolution and phylogeography of angiosperms, with special interest in the systematics and biogeography of the genus Carex (Cyperaceae).

Author contributions: M.E. and M.L. conceived the idea; T.V., S.M-B and M.L. collected the plant material; T.V., M.E. and S.M-B, carried out the lab work and analysed the data; T.V., M.E. and S.M-B led the writing and drafted the manuscript, although all authors contributed to its preparation.

255 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

Table 1. Divergence dates of clades in Carex section Glareosae. Divergence dates of clades in Carex section Glareosae presented as the posterior probability followed by the mean time to the most recent common ancestor in million years ago (Ma) and the 95% HPD interval obtained from the divergence time analysis of the combined nuclear (ITS and ETS) and plastid (5’trnK intron and rps16) regions.

Posterior Mean Min Max Clade Probability (Ma) (Ma) (Ma) Section Glareosae 0.99 6.55 2.56 10.99 C. furva + C. lachenalii + C. glareosa + C. canescens 0.98 5.5 1.84 9.17 C. lachenalii + C. glareosa 1 1.03 0.18 2.14 C. canescens 1 1.17 0.34 2.17 C. canescens from South America (2 populations) 0.85 0.05 0 0.17

256 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

LISTS OF FIGURE LEGENDS

Figure 1. Distribution map of the sampled populations of C. canescens. The shaded

region denotes the distribution of C. canescens obtained from the World

Checklist of Cyperaceae (Govaerts et al., 2014).

Figure 2. Maximum credibility clade phylogeny of the Bayesian divergence time

analysis considering Carex canescens, other related species in sect. Glareosae

and outgroups using a combined matrix of nuclear (ITS and ETS) and plastid

(5’trnK and rps16) sequences. Node bars represent the 95% highest posterior

density intervals of the divergence time estimates linked to nodes with posterior

probabilities above 0.85 (values above branches) with mean ages inferred for

clades in million years (below branches). Light grey rectangle depicts C.

canescens populations from Northern and Southern Hemisphere; dark grey

rectangle depicts two populations of C. canescens from Argentina.

Figure 3. Haplotype network of concatenated cpDNA sequences of Carex canescens

and C. canescens subsp. disjuncta. Circles represent the five haplotypes found

(H1-H5), lines represent single mutational steps, and small black circles missing

haplotypes. Number of samples per haplotype are indicated in parentheses and

abbreviations after the names correspond to the geographical regions of the

world (Brummitt, 2001).

Figure 4. Scatter plots of the first three components of the Principal components

analysis depicting the position in a climate-niche space of the Carex canescens

sampled populations geographically grouped by Earth’s quadrants (North-East,

grey dots; North-West, pink; South-East, blue; South-West, red).

257 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

258 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

259 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species: biogeography of C. canescens

260 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species:______biogeography of C. canescens

Journal of Biogeography

SUPPORTING INFORMATION

Two independent dispersals to the Southern Hemisphere to become the most widespread Carex bipolar species: biogeography

of C. canescens (Cyperaceae)

Tamara Villaverde, Marcial Escudero, Santiago Martín-Bravo and Modesto Luceño

Appendix S1. Studied material of Carex canesens, related species and outgroups; molecular characteristics of the amplified regions; results from the Principal Components Analysis of 19 bioclimatic variables from the WorldClim database; description of bioclimatic variables; and AUC values from MAXENT analyses.

261 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species:______biogeography of C. canescens

Table S.1: List of material studied of species from sect. Glareosae (Carex canescens, C. canescens subsp. disjuncta, C. glareosa, C. lachenalii, C. furva, C. arcta) and the outgroups (C. macloviana, C. paniculata subsp. paniculata, C. remota and C. maritima) including population code, coordinates, voucher information and Genbank accessions for markers used for molecular studies. Population codes correspond to geographical regions of the world (Brummitt, 2001) and population number. Species Population Latitude Longitude Voucher Genbank Accesion code (ITS, ETS, 5’trnK intron and rps16 intron) C. canescens AGS_1 -50.483197 -72.874399 Argentina: Santa Cruz, Los Glaciares Forthcoming National Park. 20-I-2010. J. Starr P14-4 10018 & T. Villaverde (UPOS3935) C. canescens AGS_2 -49.205715 -72.955547 Argentina: Santa Cruz, Los Glaciares Forthcoming National Park. 21-I-2010. J. Starr 10022/ P18-1 & T. Villaverde (UPOS3939) C. canescens AGS_3 -42.97121 -71.582108 Argentina: Chubut, Los Alerces National Forthcoming Park. 31-I-2010. J. Starr P24-1 10029 & T. Villaverde (UPOS3946) C. canescens AGS_4 -54.8333333 -68.5 Argentina: Tierra del Fuego, Ushuaia, Tierra Forthcoming del Fuego National Park. 08-I-2010. S. Martín-Bravo 7SMB10, P. Vargas & M. Luceño (UPOS4237) C. canescens AGS_5 -41.2579 -71.679915 Argentina: Río Negro, Nahuel Huapi Forthcoming National Park. 2-II-2010. J. Starr JS10032 P27-8 & T. Villaverde (UPOS3947) C. canescens AGS_6 -54.132279 -68.068818 Argentina: Tierra del Fuego, Río Grande, Forthcoming Ona River. 17-I-2010. S. Martín-Bravo 49SMB10, P. Vargas, M. González & M. Luceño (UPOS4282) C. canescens AGS_7 -54.793811 -67.642027 Argentina: Tierra del Fuego, between Forthcoming Ushuaia and Tolhuin, Rancho Hambre. 9-I- 2010. S. Martín-Bravo 15SMB10, P. Vargas,

262 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species:______biogeography of C. canescens

M. González & M. Luceño (UPOS4245)

C. canescens ASK_1 62.322317 -150.094549 USA: Alaska, Alaska Range District, Peters Forthcoming Hills Mountains. 17-VII-1980. V. Siplivinsky 666 (CAN453336) C. canescens AUT_1 47.220106 11.629783 Austria: Tirol, Ötztaler Alpen, Sölden. 11- Forthcoming VII-1993. E. Vitek 33-11, A. Blab & G. Dietrich (NSW815997) C. canescens BEL_1 50.253394 6.120001 Belgium: Hohes-Vann Park, between Eupen Forthcoming and Mönschav. 29-VI-2008. S. Martín- Bravo 88SMB08 & M. Escudero (UPOS3458) C. canescens BUL_1 42.081017 23.903942 Bulgary: Rhodopians, between Belovo and Forthcoming Jakoruda. 9-VII-2010. P. Jiménez-Mejías 147PJM10, R. Jiménez Mejías & S. Jiménez Mejías (UPOS4087) C. canescens CAL_1 38.8715 -120.113116 USA: California, El Dorado Co, El Dorado Forthcoming National Forest, Grass Lake. 23-VII-1987. J. Pykälä, D. H. Norris & H. Toivoinen 2922 (BM s.n.) C. canescens CHM_1 43.913021 125.736503 China: Jilin. J. Jung 1007076 (AJOU) JX644817

C. canescens CLS_1 -53.395722 -71.126564 Chile: Region XII of Magallanes and Forthcoming Chilean Antarctic, Brunswick Peninsula, gate of Laguna Parrilar Forest Reserve. J. Starr P13-7 10017 & T. Villaverde (UPOS3934) C. canescens CLS_2 -54.140305 -68.845772 Chile: Region XII of Magallanes and Forthcoming, Chilean Antarctic, Big Island of Tierra del EU541865(ITS), Fuego. 27-XII-2005. M. Luceño 17905ML

263 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species:______biogeography of C. canescens

& R. Álvarez (UPOS0212) EU541834 (rps16),

C. canescens FIN_1 67.906062 24.486169 Finland: Pallas-Ounastunturi National Park. Forthcoming, 4-VIII-2005. M. Luceño 2005ML & M. EU541833 (rps16), Guzmán (UPOS314) C. canescens GER_1 51.764009 13.557863 Germany: Brandenburg. VI-1903. R. Gross Forthcoming s.n. (NSW816004) C. canescens GNL_1 66.98333333 -52.3166667 Denmark: Greenland, Ikertoq, Maligiaq, Forthcoming Itivneq. 26-27-VII-1978. C. Bay 78-1623, B. Fredskild, S. Hanfgarn & P. F. Moller (CAN488181) C. canescens GNL_2 60.003958 -44.336017 Denmark: Greenland, Qassiarsuk, Forthcoming Tassiussaq fjord. 4-VIII-2007. M. Luceño 3207ML & M. Guzmán (UPOS4471) C. canescens GNL_3 60.947449 -46.613291 Denmark: Greenland, Qaleragdlit fjord. 5- Forthcoming VIII-2007. M. Luceño 4407ML & M. Guzmán (UPOS4481) C. canescens ICE_1 65.02468333 -19.1181972 Iceland: between Storaborg and Mosfell. 6- Forthcoming, VIII-2006. M. Luceño 3206ML GQ469855 (trnk), (UPOS1941) EU541836 (rps16), EU541867(ITS)

C. canescens IRK_1 57.16667 104.55 Russia: Siberia, Region of Lake Baikal, Forthcoming Valley of Korolok. 7-VII-1979. H. H. Iltis, J. C. Coffey & M. F. Denton 504 (NYBG s.n.) C. canescens IRK_2 51.723501 103.341859 Russia: Irkutskaya oblast, Valley of Forthcoming Bystraya River, west end of Lake Baikal. 14- VII-1979. H. H. Iltis, J. C. Coffey & M. F. Denton 833 (NYBG s.n.)

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C. canescens KAM_1 53.133376 159.116506 Russia: Kamchatka Peninsula, Pacific Ocean Forthcoming coast, SE of Nalychevo. 29-VII-2003. A. Farjon & G. Frisor 653 (E00270618) C. canescens KAM_2 55.913997 158.649107 Russia: Kamchatka Peninsula, Bristraya Forthcoming River, Esso. 23-VII-2003. R. K. Brummitt & G. Frisor 20883 (E00270610) C. canescens KRA_1 56.908423 96.518853 Russia: Krasnoyarsk Territory, Abansky Forthcoming District. 27-VII-1960. Pavlova & T. Litvin (NYBG s.n.) C. canescens LAB_1 53.06666667 -66.9666667 Canada: Newfoundland and Labrador, Carol Forthcoming Lake, North end. 17-IX-1953. F. Harper 4031 (CAN226641) C. canescens MAS_1 41.678902 -70.489216 USA: Massachusetts, Sandwich, Boggy Forthcoming swale. 8-VI-1932. C. A. Weatherby, U. F. Weatherby, L. B. Smith & R. C. Smith s.n. (NSW815995) C. canescens NFL_1 53.051214 -57.446935 Canada: Newfoundland and Labrador. Route Forthcoming 13 before Trans-Labrador (Hwy 510). 24- VII-2008. R. Piñeiro 80RPP08 & M. Escudero (UPOS s.n.) C. canescens NFL_2 51.421133 -55.616348 Canada: Newfoundland and Labrador, Strait Forthcoming of Belle District, on road to Goose Cove. 19- VIII-1992. A. Bouchard 92389, S. Hay & L. Brouillet (CAN566828) C. canescens NOR_1 68.160421 13.749333 Norway: Lofoten Islands, Kallen, Kabelvag, Forthcoming Svolvaer. 29-VII-2009. M. Escudero 61ME09, R. Piñeiro & M. Pimentel (UPOS s.n.) C. canescens NSW -36.3 148.3833333 Australia: New South Wales, Kosciuszko - National Park, Schlink Pass. 14-II-1985. K. L. Wilson 6151 (NSW259564)

265 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species:______biogeography of C. canescens

C. canescens NSW -36.4166667 148.35 Australia: New South Wales, Kosciuszko - National Park, near Spencer Creek. 23-I- 1975. G. Thompson 2260 (NSW656969) C. canescens NSW -36.4163889 148.4027778 Australia: New South Wales, Kosciuszko - National Park, Blue Cow. 28-I-1977. G. Thompson 2714. (NSW656966) C. canescens NSW_1 -36.432601 148.274963 Australia: New South Wales, Kosciusko, Forthcoming Karlway Morraine Creek, Mount Northcott. 15-II-1970. C. Totterdell 73 (CANB343808) C. canescens NSW_2 -36.431037 148.33854 Australia: New South Wales, Kangaroo Forthcoming Range, Charlotte. Pass to Kosciusko. 27-II- 1960. A. Gray 772 (CANB76959) C. canescens NSW_3 -36.501272 148.501272 Australia: New South Wales, Kosciusko, Forthcoming Lower Twyneham Cirque. 11-II-1962. Walker ANU-189 (CANB104756) C. canescens NWT_1 63.29613889 -129.831917 Canada: Northwest Territories, Canol Road Forthcoming (Hwy 5), MacMillan Pass. 17-VII- 2004. P. M. Peterson 18645, J. M. Saarela & S. F. Smith. (CAN590896) C. canescens NWT_2 64.900522 -125.571633 Canada: Northwest Terriotories, Mackenzie Forthcoming District, Fort Norman. 21-VII-1951. A. A. Lindsey 365a (CAN216063) C. canescens QUE_1 46.15153889 -74.5848694 Canada, Quebec. 4-VIII-1997. A. Bond s.n. AY757406 (ITS), (MTMG s.n.) AY757384 (ETS)

C. canescens ROM_1 45.602362 24.61413 Romania: Carpathians, Fagaras Mountains, Forthcoming Bâle Lake. 20-VII-2013. M. Puscas s.n. (CL s.n.) C. canescens SCO_1 56.366423 -3.217611 United Kingdom: Scotland, Perth. Glen Forthcoming Girnaig. Altt na Core Lagain. 12-VII-1989. A. C. Jermy 277960700 (BM18314)

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C. canescens SCO_2 56.670389 -5.23363 United Kingdom: Scotland, Highlands Forthcoming between Tynohumy and Glencoe. 25-VI- 2007. S. Martín-Bravo 140SMB07, P. Jiménez-Mejías & M. Luceño (UPOS s.n.) C. canescens SPA_1 42.323944 -3.534748 Spain: Burgos. Neila, Ducks Lagoon. 19- Forthcoming, VII-2000. M. Luceño 44000ML & J. Martín EU541835 (rps16) (UPOS0087) C. canescens SPA_2 41.817486 0.639162 Spain: Lérida, Baños de Tredos. 2-VIII-2004 Forthcoming J. M. Marín 7604JMM, M. Luceño & L. E. Bendrell (UPOS s.n.) C. canescens SPA_3 42.242064 -6.795057 Spain: Zamora, Peña Trevinca, Laguna de Forthcoming Cubillas. 28-VII-2007. S. Martín-Bravo 152SMB07, P. Jiménez-Mejías & I. Pulgar (UPOS s.n.) C. canescens SWE_1 67.835408 67.835408 Sweden: Torne Lappmark, Jukkasjarvi. 22- Forthcoming VII-1946. E. Nyholm s.n. (UPOS s.n.) C. canescens SWE_2 58.67097 16.938393 Sweden: Buskhyttan, Närke. 23-VI-1949. G. Forthcoming Kjellmert s.n. (UPOS s.n.) C. canescens TCS_1 40.515209 44.184896 Armenia: Mt. Aragats, Aragatsotn, Aragats. Forthcoming 28-VI-2008. G. Fayuush et al. 08-1381 (NYBG s.n.) C. canescens TCS_2 39.680281 45.731723 Armenia: Sgunik Province, between Sisian Forthcoming and Yerevan, Ughedzor Pass. 16-VI-2007. Oganesian et al. 07-0725 (NYBG s.n.) C. canescens UTA_1 40.807997 -109.511629 USA: Utah, Daggett County, Green Lake. 3- Forthcoming VI-1986. V. E. McNeilus s.n. (NSW815990) C. canescens VIC -36.851064 147.343468 Australia: Victoria, Alpine National Park, - Mt Nelse. 13-I-1982. S. J. Forbes 783, R. Adair & M. Gray. (NSW657404).

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C. canescens VIC -37.846463 146.273161 Australia: Victoria, Baw Baw Mount. 22-I- - 1969. A. S. Johnson. (NSW143407) C. canescens VIC_1 -36.405835 148.313587 Australia: Victoria, Snowfields, Bogong Forthcoming High Plains. 18-I-1988. N. G. Walsh 1939, P. S. Short & M. C. Looker (UPOS005029) C. canescens WIN_1 - - USA: Wisconsin. Hipp et al. 587 (WIS s.n.). DQ460952 (ETS) Unpublished. C. canescens YUG_1 42.709597 22.324799 Serbia: Rhodopians, Vlasina Lake, Forthcoming Vlasinsko Jezero. 20-VI-2010. P. Jiménez- Mejías 80PJM10, R. Jiménez & S. Jiménez (UPOS4722) C. canescens YUK_1 63.805779 -135.790132 Canada: Yukon, Vicinity of Halfway Lake, Forthcoming North of Mayo. 29-VI-1967. R. T. Porsild 639 (CAN312366) C. canescens MAS_1 - - USA: Massachusetts, Berkshire County, Forthcoming subsp. Thomas Pond. 9-VI-2001. T. M. Zebryk disjuncta 7312 (NSW815989) C. glareosa - - United States: Alaska, Bethel. Parker 17823 JN903115 (ETS) (MOR) C. glareosa - - Iceland: Djúpivogur, Berufjördur. 9-VIII- EU541871 (ITS) 2006. M. Luceño & M. Guzman 7206ML (UPOS1983) C. lachenalli - - Norway: Kvaenangsfjellet. 6-VIII-2005. M. EU541869 (ITS) Luceño & M. Guzman, 5305ML (UPOS354) C. furva - - Spain: Granada, Capileira, Sierra Nevada KP980522 (ITS), National Park. 08/VIII/2013. E. Maguilla & KP980331 (ETS) J. M. G. Cobos 31EMS13(1) (UPOS5132) C. arcta - - USA: Minnesota, Clearwater County, along Forthcoming County Route 39. 22-VI-1991. V. E. McNeilus 91-565 (NSW815988)

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C. macloviana Chile: between Punta Arenas and Puerto EU541862 Natales. 29-XII-2005. M. Luceño & R. Alvarez 18605ML (UPOS1804) C. macloviana - - Unitated States: Wyoming. A. L. Hipp, Hipp DQ460993 1893 (WIS) C. paniculata - - Greece, Épiro, Ioannina, Kambos Despoti. KP980427 (ITS), subsp. 23-VI-2008. M. Luceño 0808ML KP980240 (ETS) paniculata (UPOS3419) C. remota - - Montenegro, High Dinarics, Durmitor Forthcoming National Park. 17-VII-2010. P. Jiménez- Mejías 198PJM10 (UPOS4006) C.maritima - - Switzerland: Zermatt. 11-VIII-2012. M. Forthcoming Luceño & M. Guzmán 25ML12 2/2 (UPOS4997)

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Table S.2: Characteristics of the DNA regions sequenced for the complete datasets including Carex canescens, related species in section Glareosae and outrgroups. Complete dataset

5'trnKCarexF- rps16F-rpsR ITS1/ 5.8S/ ITS2 ETS1f - 18S-R 5'trnkCarexR Description Internal transcribed spacers 1 and 2 External transcribed spacer Plastid intergenic Plastid intergenic and 5.8S ribosomal RNA of ribosomal RNA spacer spacer

References Blattner (1999), White (1990) Starr et al. (2003) Escudero & Luceño Oxelman et al. (2009) (1997) Total number of sequences 56 41 47 43 in the alignment

Aligned length (bp) 613 560 659 801

Ungapped length range 505-610 214-557 439-646 422-796

% Identical sites 45.3% 79.6% 61.5% 91.5%

% Pairwise identity 94.2% 98.3% 97.6% 91.5%

Variable characters 55 69 11 21

Parsimony-informative 39 28 5 13 characters Number of informative 0030 indels Mean % G+C content 58.40% 53.50% 21.70% 25.30%

Substitution model GRT+G / HKY / GTR + G HKY + I JC + I GTR

270 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species:______biogeography of C. canescens

Table S.3: Loadings matrix obtained by the Principal Components Analysis of 19 bioclimatic variables on Carex canescens and the percentage of variance explained by each principal component. PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PC11 PC12 PC13 PC14 PC15 PC16 PC17 PC18 PC19 BIO1 -0.2 -0.4 -0.1 0.1 -0.1 0.0 -0.1 0.2 0.1 0.0 -0.2 0.6 0.0 -0.2 0.2 -0.2 0.5 0.0 0.0 BIO2 0.2 -0.3 0.2 -0.3 0.4 -0.3 0.0 -0.1 -0.2 0.5 -0.1 0.2 0.0 0.2 -0.3 0.0 0.1 0.1 0.0 BIO3 -0.2 -0.2 -0.1 -0.4 0.2 -0.6 0.1 0.1 0.3 -0.5 0.0 -0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0 BIO4 0.3 0.0 0.3 0.1 0.1 0.2 0.0 -0.1 0.1 -0.4 0.1 0.0 0.1 0.1 -0.4 -0.2 0.3 -0.6 0.0 BIO5 0.1 -0.5 0.2 0.0 0.1 0.1 0.0 0.1 -0.1 0.1 0.2 -0.6 0.0 -0.2 0.4 0.0 0.1 0.0 -0.3 BIO6 -0.3 -0.1 -0.3 0.0 -0.2 0.0 0.0 0.1 0.0 0.1 0.2 -0.4 0.1 0.1 -0.3 -0.1 0.3 0.1 0.6 BIO7 0.3 -0.1 0.3 0.0 0.2 0.1 0.0 0.0 -0.1 -0.1 -0.1 0.1 0.0 -0.2 0.4 0.1 -0.2 -0.1 0.7 BIO8 0.2 -0.2 0.0 0.3 -0.4 -0.4 0.5 -0.5 -0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 BIO9 -0.3 -0.2 -0.1 -0.2 0.1 0.2 -0.3 -0.7 -0.4 -0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 BIO10 0.0 -0.5 0.1 0.2 0.0 0.2 -0.1 0.1 0.2 -0.3 -0.1 0.1 0.0 0.2 -0.3 0.2 -0.4 0.3 0.0 BIO11 -0.3 -0.2 -0.2 0.0 -0.1 -0.1 -0.1 0.1 0.0 0.2 -0.1 0.1 0.0 0.0 0.0 0.2 -0.4 -0.7 0.0 BIO12 -0.3 0.0 0.3 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.1 0.0 0.4 -0.4 -0.2 0.6 0.2 0.0 0.0 BIO13 -0.3 0.0 0.3 -0.1 -0.2 0.0 0.1 0.1 -0.2 0.0 -0.6 -0.2 -0.1 0.5 0.2 0.1 0.2 0.0 0.0 BIO14 -0.3 0.1 0.2 0.3 0.3 0.0 0.0 -0.3 0.4 0.1 -0.1 -0.1 -0.6 -0.2 -0.1 0.1 0.1 0.0 0.0 BIO15 0.1 0.0 0.2 -0.4 -0.6 0.0 -0.4 -0.2 0.4 0.2 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 BIO16 -0.3 0.0 0.3 -0.1 -0.2 0.0 0.1 0.1 -0.2 -0.1 -0.2 0.0 0.0 -0.5 -0.3 -0.5 -0.3 0.0 0.0 BIO17 -0.3 0.1 0.2 0.2 0.3 0.0 0.0 -0.2 0.4 0.2 0.0 0.0 0.6 0.2 0.2 -0.3 -0.2 0.0 0.0 BIO18 -0.2 0.1 0.3 0.3 -0.1 -0.4 -0.5 0.1 -0.3 -0.1 0.3 0.1 -0.1 0.1 0.1 0.0 0.0 0.0 0.0 BIO19 -0.3 0.0 0.2 -0.2 0.0 0.3 0.5 0.0 0.0 0.1 0.6 0.2 -0.3 0.2 0.1 0.0 0.0 0.0 0.0 % 48.1% 15.7% 14.6% 10.5% 5.5% 3.0% 1.2% 0.6% 0.4% 0.2% 0.1% 0.1% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%

271 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species:______biogeography of C. canescens

Table S.4: Bioclimatic variables used. Units of bioclimatic variables are °C × 10 for temperature (excluding BIO 4 that was calculated based on K × 10 to deal with negative temperatures) and mm for precipitation.

Bioclimatic variable Description BIO 1 annual mean temperature BIO 2 mean diurnal temperature range [mean of monthly (maximum temperature - minimum temperature)] BIO 3 isothermality (BIO2 / BIO7 x 100) BIO 4 temperature seasonality (standard deviation of monthly temperature) BIO 5 maximum temperature of the coldest month; BIO 6 minimum temperature of the warmest month BIO 7 temperature range (BIO6 - BIO5) BIO 8 mean temperature of the wettest quarter BIO 9 mean temperature of the driest quarter BIO 10 mean temperature of the warmest quarter BIO 11 mean temperature of the coldest quarter BIO 12 annual precipitation BIO 13 precipitation of the wettest month BIO 14 precipitation of the driest month BIO 15 precipitation seasonality (coefficient of variation of monthly precipitation) BIO 16 precipitation of the wettest quarter BIO 17 precipitation of the driest quarter BIO 18 precipitation of the warmest quarter BIO 19 precipitation of the coldest quarter

272 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species:______biogeography of C. canescens

Table S.5: Area under de curve (AUC) ± standard deviation (SD) and the variables that contributed the most to explain MAXENT models under WorldClim database layers and the most important when used alone analyses, for each group of Carex canescens.

Environmental variables that contributed most and C. canescens groups AUC ± SD the most important environmental variable when used alone All samples 0.890 ± 0.001 BIO1

Samples from the Northern 0.891 ± 0.001 BIO1 Hemisphere Samples from the Southern 0.992 ± 0.002 BIO6 Hemisphere Samples from the Southern 0.996 ± 0.001 BIO6 Hemisphere – only South American Samples from the Southern 0.995 ± 0.002 BIO6 Hemisphere – only Australasian

273 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex bipolar species:______biogeography of C. canescens

References

Blattner, F.R. (1999) Direct amplification of the entire ITS region from poorly preserved plant material using recombinant PCR. Biotechniques, 27, 1180–1186.

Brummitt R.K. (2001) World geographical scheme for recording plant distributions, 2nd ed. Hunt Institute for Botanical Documentation, Pittsburgh, Pennsylvania, USA.

Escudero, M. & Luceño, M. (2009) Systematics and evolution of Carex sects. Spirostachyae and Elatae (Cyperaceae). Plant Systematics and Evolution, 279, 163–189.

Starr, J.R., Harris, S.A. & Simpson, D.A. (2003) Potential of the 5 and 3 ends of intergenic spacer (IGS) of rDNA in the Cyperaceae: new sequences for lower-level phylogenies in sedges with an example from Uncinia Pers. International Journal of Plant Sciences, 164, 213–227.

White, T.J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: A guide to methods and applications (ed. by M. Innis, D. Gelfand, D. Sninsky, and T. White), Academic Press, Orlando, Florida, pp. 315–322.

274 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex______bipolar species: biogeography of C. canescens

Journal of Biogeography

SUPPORTING INFORMATION

Two independent dispersals to the Southern Hemisphere to become the most widespread

Carex bipolar species: biogeography of C. canescens (Cyperaceae)

Tamara Villaverde, Marcial Escudero, Santiago Martín-Bravo and Modesto Luceño

Appendix S2. Distribution map of Carex canescens, phylogeny of C. canescens, boxplots

of the most important variables from the bioclimatic PCA and ecological niche models of

Carex canescens.

Figure S.1. Distribution map of the sampled populations of C. canescens obtained from

Global Biodiversity Information Facility data portal (http://www.gbif.org/, downloaded 12

February 2015; black dots). The grey region denotes the distribution of C. canescens

obtained from the World Checklist of Cyperaceae (Govaerts et al., 2014).

Figure S.2. Majority rule (50%) consensus tree derived from the Bayesian analysis of

Carex canescens and the related species in sect. Glareosae inferred from the combined

nuclear (ITS and ETS) and plastid (5’trnK and rps16) matrix. Carex macloviana, C.

remota, and C. paniculata were used as outgroups. Numbers above and below of the

branches represent the Bayesian posterior probability (>0.9 PP) and bootstrap (>60% BS)

values of the Maximum likelihood analysis, respectively. A grey rectangle highlights the C.

canescens samples of the Southern Hemisphere. Abbreviations after the names correspond

to the geographical regions of the world (Brummitt, 2001) and to the population number.

275 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex______bipolar species: biogeography of C. canescens

Figure S.3. Boxplots of the most important variables in principal components analysis

using populations from the Northern Hemisphere (NH), Australia (SH_AU) and South

America (SH_SA) obtained from Global Biodiversity Information Facility data portal

(http://www.gbif.org/, downloaded 12 February 2015).

Figure S.4. Ecological niche models of Carex canescens geographic groups. Projections of

the model to the current climatic conditions (~1950–2000). Colours correspond to habitat

suitability with values ranging from 0 to 1 (from white to red, respectively). (a) C.

canescens; (b) C. canescens populations from the Northern Hemisphere; (c) C. canescens

populations from the Southern Hemisphere.

276 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex______bipolar species: biogeography of C. canescens

277 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex______bipolar species: biogeography of C. canescens

278 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex______bipolar species: biogeography of C. canescens

279 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex______bipolar species: biogeography of C. canescens

280 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex______bipolar species: biogeography of C. canescens

281 Chapter 5. Two independent dispersals to the Southern Hemisphere to become the most widespread ______Carex______bipolar species: biogeography of C. canescens

References

Brummitt, R.K. (2001) World geographical scheme for recording plant distributions, 2nd ed. Hunt Institute for Botanical Documentation, Pittsburgh, Pennsylvania, USA.

Govaerts, R., Koopman, J., Simpson, D., Goetghebeur, P., Wilson, K., Egorova, T. & Bruhl, J. (2014) World Checklist of Cyperaceae. Facilitated by the Royal Botanic Gardens, Kew. Published on the Internet; http://apps.kew.org/wcsp/ (Retrieved 2014-11-28).

282 Chapter 6

General discussion and conclusions

283

284 ______Chapter 6. General discussion and conclusions

Carex arctogena is a bipolar species

Carex capitata and C. arcotgena are found to be morphologically different and populations

from South America correspond to C. arctogena. Therefore, Carex antarctogena should be

treated as a synonym of C. arctogena. Morphological variability, which also corresponds

with geographical distribution and ecological differentiation (Chapters 2 and 3), was found

within populations from western North America and we suggest the description of one

species and two subspecies: Carex cayouetteana, C. cayouetteana subsp. bajasierra and C.

cayouetteana subsp. altasierra. These three new taxa diverged during the Pleistocene [0.81

million years ago (Ma); 95% highest posterior density (HPD). 0.19- 1.66 Ma], a time of

climatic changes in the Northern Hemisphere that affected plant distributions in both North

America and Eurasia (e.g. Tremblay & Schoen, 1999; Abbott et al., 2000; Alsos et al.,

2005; Eidesen et al., 2007).

Contrary to what might have happened in Europe, where mountain chains are East-West

oriented, the advance and the retreat of the ice sheet during glacial periods could have had a

less severe effect shifting species distribution in latitude during climate changes, due to the

continuous mountains chains in a North-South direction (Albach et al., 2006) and to the

vast area available below the ice sheet in North America. Carex cayouetteana subsp.

bajasierra might have adapted to boreal habitats where it occurs now, whilst Carex

cayouetteana subsp. altasierra might have been isolated in tundra habitats in southern

California.

Population fragmentation during glacial periods may have led to the formation of new

species (Abbott & Brochmann, 2003) and their small population sizes could have led to the

285 ______Chapter 6. General discussion and conclusions

acquisition of morphological traits at a faster rate than in large populations (Vanderpoorten

& Shaw, 2010). A similar pattern appears to have occurred in the C. aquatilis Wahlenb. -

C. lenticularis Michx. group (section Phagocystis, Dragon & Barrington, (2009) where

species ecological specialization and the Quaternary geological history of North America,

split the group in different lineages

The lack of unique characters in chloroplast data set of the new taxa here described could

be due to insufficient phylogenetic signal or incomplete lineage sorting, as suggested for

other Carex species (e.g. Hipp et al., 2006; King & Roalson, 2008; Roalson & Friar, 2008).

Likewise, a multidisciplinary study of the C. backii complex (sect. Phyllostachyae),

revealed that it was composed by three different species (Saarela & Ford, 2001), C.

saximontana Mack., C. cordillerana Saarela & Ford and C. latebracteata Waterf. Although

all quantitative characters measured in the morphological study of C. saximontana, C.

cordillerana overlapped, anatomic, micromorphologic and phytogeographic characters

showed enough differences to recognize the three taxa at the species level which was later

supported by molecular studies (Ford et al., 2009). As the three new taxa here described are

not part of independent lineages, we suggest that studies at the population level could help

to reveal its genetic differentiation.

Long-distance dispersal as the main factor underlying bipolar disjunctions in Carex

Two hypotheses can be clearly rejected for all bipolar Carex species: vicariance and

convergent evolution. The vicariance hypothesis can be rejected because the fragmentation

of the trans-tropical highland bridges during the Mesozoic Era (from the early Jurassic, 195

286 ______Chapter 6. General discussion and conclusions

Ma; Scotese et al., 1988; Figure 3 in Chapter 1) is older than the time of divergence of the

Cyperaceae family (82.6 Ma, 95% highest posterior density, HPD: 75.9–85.6 Ma; Escudero

& Hipp, 2013). Therefore, bipolar disjunctions in Carex are not due to vicariance as the

result of area fragmentation. Moreover, the same reasoning may be applied for all the

remaining bipolar species, as the divergence time estimations for their respective families

are younger than the early Jurassic (see Table 1), except for Huperzia selago, which its

family (Lycopodiaceae) diverged during the late Devonian – early Carboniferous (329.1 –

372.9 Ma).

287 ______Chapter 6. General discussion and conclusions

Table 1. Divergence time estimated for the families including bipolar species and

corresponding reference.

Family Epoch Reference Lycopodiaceae Late Devonian – Early Wikström & Kenrick (2001) Carboniferous (329,1 – 372,9 Ma) Hymenophyllaceae Middle Jurassic (ca. 155,5 – 191,7 Hennequin et al. (2008) Ma) Polygonaceae Early to late Cretaceous (ca. Schuster et al. (2013) 103,1– 125,0 Ma) Caryophyllaceae Early Cretaceous ( 111– 104 Ma) Winkstrom et al. (2001); Zeng et al. (2014) Ranunculaceae Late Jurassic - Early Cretaceous Wikström et al. (2001); Zeng et al. (131– 147 Ma) (2014) Plantaginaceae Miocene (7 – 11 Ma) Li et al. (2014)

Plumbaginaceae Pliocene - Quaternary (5 - 2,5 Lledó et al., 2005 Ma) Gentianaceae Late Cretaceous (83– 89 Ma) Wikström et al. (2001); Zeng et al. (2014) Scrophulariaceae Miocene (ca. 10 Ma) Verboom et al. (2009) Juncaginaceae Miocene (ca. 10 Ma) Janssen & Bremer (2004); von Mering, (2013) Poaceae Early Cretaceous (113– 117 Ma) Janssen & Bremer (2004) Cyperaceae Late Cretaceous (65.5–55.8 Ma) Escudero & Hipp (2013)

All Carex bipolar species share an immediate common ancestor for Northern and Southern

Hemispheres populations (Escudero et al., 2010a). Carex microglochin was retrieved as

polyphyletic in nuclear and plastid analyses (Escudero et al., 2010): two samples from

Chile appeared together with C. pulicaris and C. macrostyla in a strongly supported and

phylogenetically distant clade from the remaining five samples (Iceland, Norway,

Greenland), which grouped with Uncinia lechleriana, C. pauciflora, and C. curvula. Carex

microglochin samples from the Northern and Southern Hemisphere were also studied by

288 ______Chapter 6. General discussion and conclusions

Starr et al. (2008) and were retrieved as polyphyletic too, which leaded these authors to the

conclusion that it may be a consequence of hybridization, pseudogenes, or other factors.

Later on, it has been known that those populations from Chile were misidentified samples

of C. camptoglochin V. I. Krecz. (M. Luceño, personal communication). Carex

microglochin and C. camptoglochin differ both morphologically and ecologically, growing

sympatrically in both the northern and southern parts of South America, but C.

camptoglochin is restricted to South America and therefore is not a bipolar species

(Wheeler & Guaglianone, 2003). Therefore, we can reject the convergent evolution

hypothesis for all Carex bipolar species.

From the results of our work, the only hypothesis that cannot be rejected for any of the

bipolar Carex species is long-distance dispersal. Although long-distance dispersal

hypohtesis is not falsifiable for any taxon (i.e. distribution of all species could be explained

by dispersal), studies with phylogenetic reconstruction coupled with divergence time

estimation analyses have indicated that long-distance dispersal has played a major role in

shaping species distributions with respect to what previously thought (de Queiroz, 2005;

Michalak et al., 2010; Renner et al., 2010). Even traditional, paradigmatic examples of taxa

with a vicariant distribution, such as Araucaria or Nothofagus have been demonstrated

achieved part of its distribution by long-distance dispersal (Swenson et al., 2001; Cook &

Crisp, 2005). Therefore, although long-distance dispersal hypothesis is not rejectable,

vicariance hypothesis can be falsified and thus, dispersal would be support by default (de

Queiroz, 2005). Many studies comparing multiple taxa do not support vicariance

hypothesis for their disjunct distributions but dispersal [e.g. in South Africa (Galley &

289 ______Chapter 6. General discussion and conclusions

Linder, 2006); in New Zealand (Winkworth et al., 2005; Waters & Craw, 2006); in

Madagascar (Yoder and Nowak, 2006)].

Long-distance dispersal may have been either achieved by mountain-hopping (‘stepping

stones’) or by a direct event (a ‘giant leap’). This could have occurred during some of the

last cold periods at the end of the Pliocene (5.3 – 2.6 Ma) or in the Pleistocene (2.6 – 0.01

Ma), when the polar regions in both hemispheres recurrently expanded (Raven, 1963; Ball,

1990), or even at present times. Divergence times of the clades where the bipolar species

studied by us are found fall in the late Miocene to early Pleistocene in C. arctogena (5 Ma;

95% HPD: 2.01 – 8.03 Ma); middle–late Pliocene to early–middle Pleistocene in C.

maritima (0.23 Ma; 95% HPD: 0.03– 0.51 Ma); and during the Pleistocene in C. canescens

(1.17 Ma; 95% HPD: 0.34– 2.17 Ma; Figure 1). These ages are therefore embraced within

periods in which major climatic changes expanded polar regions in both hemispheres.

290 ______Chapter 6. General discussion and conclusions

Figure 1. Combined maximum-credibility-clade partial phylogenies from the Bayesian

divergence-time analysis of the studied Carex bipolar species (see Chapters 3, 4 and 5 for

details). Node bars represent the 95% highest posterior density intervals for the divergence-

time estimates of nodes and vertical color bars highlight these intervals for species

divergence ages (C. arctogena, blue; C. canescens, green; and C. maritima, yellow).

291 ______Chapter 6. General discussion and conclusions

Geological and climatic changes since the Miocene that allowed Northern and Southern

Hemisphere connections for bipolar species

Since the late Cenozoic, and particularly since the Miocene (23 Ma) the climate has

suffered significant variations related to various causes such as continental displacement

due to plate tectonics, modification on the concentration of greenhouse gases in the lower

atmosphere and changes in astronomical parameters (e.g. eccentricity of the Earth orbit,

obliquity of the planetary axis and equinoctial precession; Rabassa et al., 2005). All these

changes led to climatic cycles of cold and warm periods and the development of planetary

ice ages since the Miocene (Rabassa et al., 2005; Rabassa & Coronato, 2009).

Geologically, different uplifts occurred since the Miocene. In Australasia, there is a system

of island arcs that goes from the eastern side of Australia to northern Australia and New

Guinea and links the southwestern Pacific region with Indonesia. These mountain belts are

the result of multiple collisions during the last 40 (million years) My, and particularly

during the last 25 My, and created a range of ca. 15.000 km with summits well above 4000

m (Figure. 2; Audley-Charles, 1991).

292 ______Chapter 6. General discussion and conclusions

Figure 2. Island-arc systems linked to New Guinea and associated basins. Abbreviation:

S.F.Z., Sorong fault zone. Figure taken from Audley-Charles (1991).

In South America, the uplift of the central Andes (Earth’s second largest mountain belt) is

the result of an oceanic lithosphere subducted beneath continental lithosphere that started

its uplift 70-50 Ma (McQuarrie et al., 2005). In this area, there is the drained Altiplano

basin flanked by the Western and Eastern Cordilleras, with peak elevations exceeding 6000

m. Since the beginning of the Miocene (23 Ma), the uplift of the Andes changed

dramatically the South American continent (e.g. it formed the only barrier to atmospheric

circulation in the Southern Hemisphere; Lenters et al., 1995). The central Andean plateau

probably started its elevation 20 Ma (McQuarrie et al., 2005), during the early Miocene, at

an age that coincides with the diversification of the first montane plant and animal genera

(reviewed in Hoorn et al., 2010). Sedimentology and carbon istoypes in the Altiplano and

293 ______Chapter 6. General discussion and conclusions

Eastern Cordillera suggest that the central Altiplano became more arid 10-6 Mya (middle-

late Miocene, Quade et al., 2007), and it correspond to a time when the plateau experienced

other uplifts (Garzione et al., 2008; Hoorn et al., 2010; Figure 3-F). During this time, an

extensive migration occurred (known as the Great American Biotic Interchange, GABI) and

the new montane habitats in the Andes were colonized by taxa from North America (Hoorn

et al., 201). Therefore, since the divergence of the bipolar species studied, there have been

different mountain ranges allowing the connection between the Northern and Southern

Hemisphere.

294 ______Chapter 6. General discussion and conclusions

Figure 3. Paleogeographic maps from 65 to < 2.5 Mya (modified from Hoorn et al.,

2010). (A) Amazonia once extended over most of northern South America. Breakup of the

Pacific plates changed the geography and the Andes started uplifting. (B) The Andes

continued to rise with the main drainage toward the northwest. (C) Mountain building in

the Central and Northern Andes (ca. 12 Ma). (D) Uplifts of the Northern Andes. (E)

Closing of Panama Isthmus and start of GABI. (F) Quaternary.

295 ______Chapter 6. General discussion and conclusions

Direct long-distance dispersal vs. mountain-hopping

Since the Miocene, the Earth has been going through several ice ages that might have

affected plant distributions. Two different mountain ranges connect the Northern and

Southern Hemispheres, allowing migration of cold-adapted plant species by mountain-

hopping. First, a route connecting North and South America through the American

cordillera has been in place since the late Miocene (ca. 12 Ma; Smith, 1986). Then, a

gradual uplift of the cordillera during the late Pliocene created the high mountainous

environment with a much colder climate later on during the Pleistocene compared to that

incident today at the same latitudes and elevations (van der Hammen, 1974). This route has

acted as a corridor for the dispersal of different organisms (e.g. Moreno et al., 1994;

Antonelli & Sanmartín, 2011). Second, the mountain uplift in Malaysia in the Miocene-

Pliocene (10 Ma; Sanmartín & Ronquist, 2004) and the gradually cooler climate in both

hemispheres at that time (Scotese, 2004) could have facilitated the dispersal of cold-

adapted species into the Southern Hemisphere (Smith, 1981).

For Carex species, no ecophysiological adaptations to cross the short-day conditions of the

tropical alpine environment seem to be necessary (Heide, 2002), but we are not aware of

any published fossil records or any other evidence for the occurrence of C. arctogena, C.

canescens or C. maritima in many vast areas between northern North America and southern

South America, or between Eurasia and Australia. If C. arctogena or C. canescens had

migrated to South America and/or Australia by the slow and gradual means predicted by

mountain-hopping, we would expect that such a process would have left a trace of genetic

differences in the plastid loci of populations from both hemispheres (Brochmann et al.,

2003; Scotland, 2011). Although we cannot completely reject the mountain-hopping

296 ______Chapter 6. General discussion and conclusions

hypothesis, the absence of genetic variability between populations of C. arctogena and C.

canescens from both hemispheres and their recent diversification times fit better with a

recent and direct long-distance dispersal. In congruence, direct long-distance dispersal has

been shown to be remarkably frequent in some other species of Cyperaceae (e.g. Viljoen et

al., 2013).

In the case of C. maritima, its genetic structure allows us to support both a direct long-

distance dispersal or mountain-hopping. In the case of direct long-distance dispersal to the

Southern Hemisphere, it might be an older event of dispersal than in C. arctogena and C.

canescens due to the detected genetic differences between Hemispheres. It could have

arrived to South America and differentiated genetically with subsequent northward or

southward colonization along the Andes. In the case of mountain-hopping migration, it

might have occurred since the early Pleistocene, with a subsequent extinction of most of the

northern South American intermediate populations.

In conclusion, our results seem to suggest that C. arctogena and, maybe, C. canescens

could have achieved its current bipolar distribution through direct long-distance dispersal.

For the particular case of C. maritima, we conclude that the bipolar disjunction could have

been originated either by mountain-hopping along the American cordillera or through direct

long-distance dispersal.

North to South long-distance dispersal

The patterns of genetic diversity found in the three bipolar Carex species studied points to

North-to-South as the prevalent direction in bipolar long-distance dispersals between

297 ______Chapter 6. General discussion and conclusions

Hemispheres. The most widely distributed haplotypes were always found throughout the

Northern Hemisphere and comprised the highest number of haplotype connections, which

implies under the coalescent theory that they amount to the ancestral haplotypes (Posada &

Crandall, 2001; Figure 4). Moreover, the sections to which these bipolar species are

ascribed (sect. Capituligereae, C. arctogena; sect. Glareosae, C. canescens; sect. Foetidae,

C. maritima) have the majority of their species distributed in the Northern Hemisphere, a

fact that supports a northern origin and subsequent North-to-South long-distance dispersal.

298 ______Chapter 6. General discussion and conclusions

◄ Figure 4. Combined TCS haplotype networks of concatenated cpDNA sequences of the

studied bipolar Carex species and their sister species: (a) C. arctogena. Shaded and dashed

squares represent the geographical distributions of lineages; (b) C. maritima; and (c) C.

canescens; (see Chapters 3, 4 and 5 for details). Circles represent haplotypes, lines

represent single mutational steps and small black circles are missing haplotypes. Circle

shades indicate species, and numbers in parentheses indicate the number of samples per

haplotype.

Accordingly to our results, Northern to Southern Hemisphere long-distance dispersal is a

very frequent pattern of dispersal in plant species (e.g. Vargas et al., 1998; Vijverberg et al.,

1999; Yokoyama et al., 2000; Clayton et al., 2009; Escudero et al., 2009; Schaefer et al.,

2009; Wen & Ickert-Bond, 2009; Emadzade et al., 2011; Popp et al., 2011; Banasiak et al.,

2013; Lewis et al., 2014) and even in other bipolar species such as the lichen Cetraria

aculeata (Fernández-Mendoza & Printzen, 2013). However, other plant genera present the

opposite direction of dispersal. For example, the centre of origin of genus Larrea

(Zygophyllaceae) is located in South America and this genus was inferred to have migrated

to North America during the late Neogene by long-distance dispersal, using way stations in

Peru and Bolivia and probably mediated by birds (Lia et al., 2001). Likewise, the

Rubiaceae family was inferred to have migrated from South to North America during the

late Palaeocene–early Eocene using land bridges (Antonelli et al., 2009), as

Hoffmannseggia glauca (Fabaceae) via birds during the late Miocene or later (Simpson et

al., 2005).

299 ______Chapter 6. General discussion and conclusions

Means of dispersal

Wind and water dispersal

The utricle surrounding Carex fruit can show some features dispersal, as seen in the

inflated utricles of C. physodes for wind-dispersal (Egorova, 1999); in C. baccans, whose

red utricles are attractive to birds; or in C. paniculata, whose corky pericarp allows water

dispersal (reviewed in Allessio Leck & Schütz, 2005). One of the Carex bipolar species, C.

microglochin, has spikes with finely acute perigynia that reflex at maturity and are easily

detached, a characteristic that is suggested to facilitate animal dispersal (Savile, 1972). A

similar device has evolved independently in another bipolar species, Triglochin palustris

(Juncaginaceae; Savile, 1972).

However, with the exception of the above mentioned characteristics of the bladder-like

small utricles, Carex generally lack any obvious morphological features for dispersal by

abiotic or biotic forces. With the exception of C. microglochin, none of the bipolar Carex

species displays standard morphological syndromes for long-distance dispersal (as

described by Higgins et al., 2003). In fact, long-distance dispersal of seeds (> 100 km)

might not be necessarily driven by those vectors inferred from plant morphology; they are

usually associated with stochastic events (unusual behaviour of regular events or a

combination of vectors; Nathan et al., 2008).

Some dispersal agents are typically involved in long-distance dispersal, such as birds, ocean

or wind currents (Nathan et al., 2008; Gillespie et al., 2012). Considering the extreme

dispersal distance together with the shape and structure of the bipolar Carex propagules, we

consider that its dispersal was more likely to have been mediated by migratory animals than

300 ______Chapter 6. General discussion and conclusions

by wind or ocean currents, which seem insufficient for such enormous task. Besides, there

are no wind or oceans currents connecting Northern a Southern Hemispheres. Both wind

and ocean currents have opposite directions when approaching the Equator (Hyeong et al.,

2005); thus, if propagules have been transported by wind or ocean currents, they would

have to make a stop at low latitudes and then have been transported again into the other

hemisphere. Besides, successful oceanic dispersal is influenced by a complex interaction

between ocean dynamics and geomorphology at past and present times, together with the

ability of plants for survival during transportation (Gillespie et al., 2012). Although it

cannot be discarded, it seems an extremely unlikely event for bipolar plant dispersals.

Nonetheless, wind and ocean currents have been suggested to be responsible for plant

migration within hemispheres (e.g. Brooker et al., 2001; Brochmann et al., 2003; Renner,

2004; Alsos et al., 2009; Gillespie et al., 2012).

Bird dispersal

Some birds which migrate from North America to temperate zones of South America have

already been pointed out as the most likely dispersal agents of the several disjunct plant

groups (Cruden, 1966; and references therein; Popp et al., 2011; Lewis et al., 2014). Carex

seeds have been reported to be intact after transport by birds (Mueller & van der Valk,

2002), and arrivals of Carex species to newly formed islands have predominantly been

reported to happen when seeds were embedded in mud attached to birds’ feet or else when

eaten and carried inside by birds (Carlquist, 1967). Therefore, birds seem to be playing an

important role in Carex dispersal.

301 ______Chapter 6. General discussion and conclusions

Bird-mediated direct long-distance dispersal from North America to South America has

already been used to explain a bipolar disjunction in crowberries (Empetrum; Popp et al.,

2011). Most migratory birds that disperse seeds live in temperate and boreal regions

(Wheelwright, 1988). For birds to act as vectors for seed dispersal by endo- or

ectozoochory, the seeds must have morphological features for association with these

animals, and must be able to maintain their viability after intestinal transit to allow for

establishment in new environments (Gillespie et al., 2012). However, it has been proven

that Carex species are transported even in the lack of obvious morphological features for

zoochorous dispersal (Carlquist, 1967), as it happens to the three bipolar species studied

here.

Other structures or features that are not directly related with dispersal syndromes may be

involved, including anatomical features such as deposits of silica in the pericarp that harden

seeds (Graven et al., 1996; Prychid et al., 2004). These silica deposits could protect seeds

when passing through birds’ alimentary tracts (Graven et al., 1996) but could also make the

seeds as hard as pebbles and useful for grinding other organic material in bird gizzards.

Carex fruits could therefore be doubly preferred by birds – both as nourishment and as

gastroliths (Alexander et al., 1996). Some birds from North America, such as the pectoral

sandpiper, Calidris melanotos (Holmes & Pitelka, 1998), and the lesser yellowlegs, Tringa

flavipes (Tibbitts & Moskoff, 1999), are known to feed in sedge meadows before migrating

southwards to their wintering grounds in South America. Their breeding ranges closely

match the current distribution of Carex bipolar species in North and South America.

Although current bird migratory patterns do not necessarily coincide with past migrations,

these observations suggest that the bipolar disjunction in Carex species may have

302 ______Chapter 6. General discussion and conclusions

originated via bird-mediated long-distance dispersal. Additionally, dispersal may occur

through accidental displacement – vagrant birds or migrants, such as those flying to

Australia or New Zealand, deviating widely from their normal route (Battley et al., 2012).

With satellite telemetry, Gill et al. (2009) recorded transoceanic flights of bar-tailed

godwits (Limosa lapponica baueri) from Alaska to New Zealand and showed that they can

fly 10,153 km (± 1043 SD) non-stop in 7.8 days (± 1.3 SD). This extraordinary flight,

combined with species that can be preferentially chosen for fuel, could help species to

achieve a bipolar distribution by means of direct long-distance dispersal. Therefore, we

consider it plausible that bipolar Carex species could have acquired its bipolar distribution

by means of bird-mediated dispersal.

Human introductions

Six out of the 30 bipolar species are confirmed introductions or suspicious of being

introduced in the Southern Hemisphere (Table 1). Carex maritima is the only species

studied here that molecular data have been enough to prove a non-anthropochorus origin,

since Southern Hemisphere populations diverged during the early-middle Pleistocene. For

C. canescens, the weakly supported clade of two Argentinian samples, do not allow us to

reject that hypothesis; neither for C. arctogena, whose Southern Hemisphere populations

are genetically identical to the Northern Hemisphere ones. In these cases, species’

adaptation to local environmental conditions, biotic interactions and demographic processes

of these species would all have been established relatively quickly (Theoharides & Dukes,

2007). Populations of C. arctogena and C. canescens in Patagonia occur in well-conserved

303 ______Chapter 6. General discussion and conclusions

habitats and most are only accessible on foot. Specimens of C. arctogena from Patagonia

are few in the South American herbaria BA, BAA, BAB, BCRU, HIP and SI, with some

dating to the late 1880s, when the human influence in the southernmost parts of South

America was very limited. Although we cannot strongly rule out an anthropogenic

introduction of neither of these two species to South America, it seems very unlikely.

Unusual behaviour of vectors

A typical seed morphology has traditionally been associated to a particular dispersal vector,

(haplochory; i.e. dispersal mediated by a single standard dispersal vector; Nathan et al.,

2008); however, there is now more evidence of dispersal mediated by more than one vector

(i.e. polychory; reviewed in Nathan et al., 2008). One example of this shift is the dispersal

of Taraxacum officinale, a typical wind-dispersed species whose hairy seeds have a half-

time buoyancy of 2.57 days in water (Boedeltje et al., 2003) and potential for ectozoochory

(Tackenberg et al., 2006). When dandelion seeds are wind dispersed, they go away from the

mother plant around 2.15 meters (Soons & Ozinga, 2005) whereas when they are water-

dispersed or animal-dispersed, they can fairly increase that distance and hence, the potential

for long-distance dispersal by other vectors. In fact, species with small seeds such as those

of the dandelion and/or from water-influenced habitats are often highly dispersible taxa

(Mcglone et al., 2001). Therefore, species can be dispersed longer distances with

unpredicted or non-associated vectors than with vectors directly associated to its dispersal

syndromes. This could also be the case in arctic plant species, that have been demonstrated

to migrate enormous distances despite of the lack of specific syndromes (Abbott et al.,

2003). Thus, we suggest that relatively unspecialized structures for dispersal might play a

role in the distribution of these bipolar Carex species and we regard the hypothesis of non-

304 ______Chapter 6. General discussion and conclusions

standard vector-mediated dispersal, either by abiotic or biotic forces, as a possible

explanation of their bipolar disjunction.

Nonetheless, rare processes or unusual behaviour of vectors have been inferred to have

dispersed seeds over long distances (Higgins & Richardson, 1999; Nathan et al., 2002,

2008; Higgins et al., 2003). In fact, long-distance dispersal is claimed as a widespread

phenomenon in many plant species without standard morphological syndromes for long-

distance dispersal (Carlquist, 1967; Cain et al., 2000; Higgins et al., 2003; Alsos et al.,

2007; Dixon et al., 2009).

Successful establishment after dispersal in Carex bipolar species

To produce a disjunct distribution, long-distance dispersal has to be followed by the

establishment of a permanent population in a new area. In many cases, establishment in a

new environment - which is determined by the environment and biotic conditions of the

host community (Mitchell et al., 2006) - may be more difficult to achieve that long-distance

dispersal per se. Therefore, it is not the chance of dispersal alone but the entire colonization

process, this is, dispersal followed by establishment, which is critical for a species to

expand its geographical range.

Our results show that in the Southern Hemisphere the three Carex bipolar species studied

occupy a more restricted climatic niche than in the Northern Hemisphere. It is more

differentiated in C. maritima and C. arctogena than in C. canescens, whose Southern

Hemisphere populations fall within the general ecological conditions tolerated by the

species, which seem to be very wide. This ecological plasticity of C. arctogena and C.

305 ______Chapter 6. General discussion and conclusions

maritima or the tolerance of harsher conditions in the Northern Hemisphere than in the

Southern Hemisphere could have been a key element in their establishment after dispersal.

If these species have been successful colonizing the Southern Hemisphere, why are not they

in the same ecological niche in the Northern Hemisphere? Are there any of the Southern

Hemisphere niches available in the Northern Hemisphere? If yes, why are not they found

there? One possible reason could be found in community assemblies or competitive

interaction of those areas (Waters, 2011), which could be easier to penetrate in the Southern

Hemisphere than in potential areas in the Northern Hemisphere. Such biotic differences

could have allowed them to shift into new habitats and climate zones (Broennimann et al.,

2007) in the Southern Hemisphere. Alternatively, establishment could have taken place at a

time when both areas had similar climatic conditions. More robust conclusions could be

obtained with principal component analysis methods by measuring climatic niche shifts

using Bayesian generalized linear models (e.g. González-Moreno et al., 2014).

Intrinsic conditions for long-distance dispersal and establishment

Self-fertilization is a reproductive characteristic displayed by many species with disjunct

populations in the temperate zones (Carlquist, 1983). This attribute could play in favour of

local survival and establishment after long-distance dispersal events, given that a single

propagule of self-compatible individuals could in principle be sufficient to start a sexually-

reproducing colony (Baker, 1955). In congruence, Carex species are predominantly

monoecious and in general highly self-pollinated (Friedman & Barrett, 2009), which has

been inferred from studies based on hand pollinations, isozyme work (e.g. Ohkawa et al.,

306 ______Chapter 6. General discussion and conclusions

2000; Friedman & Barrett, 2009) and microsatellite data (e.g. Escudero et al., 2010b, 2013).

This characteristic could explain, at least in part, the often successful colonization of Carex

species after a long-distance dispersal event (Moore et al., 1971; Ball, 1990; Escudero et

al., 2009).

In addition, chromosome rearrangements have been shown to be correlated with phenotypic

differences, as well as being thought to enhance fitness in different habitats (Coghlan et al.,

2005). Specifically, high chromosome number evolution is a result of: (i) selection by

climatic regime and ecological strategies; and (ii) neutral processes such as phylogenetic

inertia or migration processes (Escudero et al., 2012, 2013). The high chromosome number

variation in some bipolar Carex species [C. canescens, 2n = 52-54, 56-58, 60, 62; C.

macloviana, 2n= 82, 82-86, 86; C. magellanica, 2n= 58, ca. 60; reviewed in Roalson et al.,

2008) might also be a result of the influence of the climatic environments at different

latitudes and distant regions. Carex arctogena (2n=50) and C. maritima (2n=60) have not

been reported to display chromosome number variation (Roalson, 2008).

307 ______Chapter 6. General discussion and conclusions

Conclusions

1. Carex capitata and C. arctogena are two different species; populations from South

America correspond to C. arctogena, and thus, Carex antarctogena is a synonym of

C. arctogena.

2. The morphological variation found in populations from Western North America

leads to the description of one new species C. cayouetteana, and two new

subspecies C. cayouetteana subsp. altasierra and C. cayouetteana subsp.

bajasierra.

3. The genus Carex is the genus with the largest number of bipolar species (six).

4. Two hypotheses can be rejected for all bipolar Carex species: vicariance and

convergent evolution. They only hypothesis that cannot be rejected is long-distance

dispersal.

5. The bipolar species studied have probably migrated from the Northern Hemisphere

to the Southern Hemisphere.

6. All bipolar Carex species studied originated from the late Miocene: C. arctogena

diverged in the late Miocene to early Pleistocene (5 Ma; 95% HPD: 2.01 – 8.03

Ma); C. maritima during middle–late Pliocene to early–middle Pleistocene (0.23

Ma; 95% HPD: 0.03– 0.51 Ma); and C. canescens during the Pleistocene (1.17 Ma;

95% HPD: 0.34– 2.17 Ma).

7. There are no genetic differences between Northern and Southern Hemispheres

populations of C. arctogena and C. canescens, which suggest a recent, direct long-

distance dispersal, probably mediated by birds.

308 ______Chapter 6. General discussion and conclusions

8. Carex maritima populations from the Southern Hemisphere were genetically and

ecologically differentiated from their northern counterparts.

9. Carex canescens have dispersed at least twice to the Southern Hemisphere, once to

Australia and once to South America.

309 ______Chapter 6. General discussion and conclusions

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