Genetic Structure and Early Effects of Inbreeding in Fragmented Temperate Forests of a Self-Incompatible Tree, Coccineum

PAULA MATHIASEN, ADRIANA E. ROVERE, AND ANDREA C. PREMOLI∗ Laboratorio Ecotono, Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, Quintral 1250, 8400 Bariloche, Argentina

Abstract: Deforestation of temperate forests has created landscapes of forest remnants in matrices of intense human use. We studied the genetic effects of fragmentation in southern Chile on J.R. et G. Forster, an early colonizing, bird-pollinated tree. We tested the hypothesis that, because of its self-incompatibility and life-history strategy, E. coccineum is less strongly affected by fragmentation. We studied the effects of reduced population size and increased isolation on population genetic structure and early performance of progeny. Samples were collected from spatially isolated trees and six fragments of differing sizes (small, 1 ha; medium, 20 ha; large, >150 ha). Based on isozyme polymorphisms we estimated parameters of genetic diversity, di- vergence, and inbreeding for adults and greenhouse-grown progeny. We also measured germination, seedling growth, and outcrossing rates on progeny arrays. Genetic variation of adults did not correlate significantly with population size, as expected, given that fragmentation occurred relatively recently. Weak effects of fragmenta- tion were measured on progeny. Only adults yielded significant inbreeding. Similar total genetic diversity was found in adults and progeny. Low but significant genetic differentiation existed among adult and progeny pop- ulations. Seedling growth correlated positively with the effective number of alleles, showing deleterious effects of inbreeding on progeny. Seeds from small fragments had the highest outcrossing rates and germination success, indicating that higher pollinator activity in such fragments reduced selfing, thereby buffering genetic erosion and maintaining adaptive variation. The effects of forest fragmentation were detectable in E. coccineum, but these effects will probably not be detrimental to the viability of remnant populations because small, fragmented populations demonstrated higher levels of gene flow and lower inbreeding than larger stands. Pioneer species that are insensitive to forest clearing may be crucial in recovery plans to facilitate the establishment of species intolerant to such disturbance.

Keywords: fragmentation, genetic diversity, outcrossing rates, population size, progeny vigor, temperate forest

Estructura Gen´etica y Efectos Tempranos de la Endogamia en Bosques Templados Fragmentados de un Arbol´ Auto Incompatible, Embothrium coccineum Resumen: La deforestacion´ de bosques templados ha creado paisajes con remanentes de bosques en ma- trices de uso humano intenso. Estudiamos los efectos gen´eticos de la fragmentacion´ en el sur de Chile sobre Embothrium coccineum J.R. et G. Forster, un arbol´ colonizador primario, polinizado por aves. Probamos la hipotesis´ que, debido a su auto incompatibilidad y estrategia de historia de vida, E. coccineum es poco afectada por la fragmentacion.´ Estudiamos los efectos de la reduccion´ del tamano˜ poblacional y del incremento del aislamiento sobre la estructura gen´etica de la poblacion´ y el vigor temprano de la progenie. Se recolectaron muestras de arboles´ espacialmente aislados y en seis fragmentos de diferente tamano˜ (pequeno,˜ 1 ha; medi- ano, 20 ha; grande, >150 ha). En base a polimorfismos de isozimas estimamos parametros´ de la diversidad

∗Address correspondence to A. C. Premoli, email [email protected] Paper submitted January 10, 2006; revised manuscript accepted May 8, 2006. 232 Conservation Biology Volume 21, No. 1, 232–240 C 2007 Society for Conservation Biology, No claim to US government works DOI: 10.1111/j.1523-1739.2006.00565.x Mathiasen et al. Genetic Effects of Fragmentation 233 gen´etica, divergencia y endogamia en adultos y progenie cultivada en invernadero. Tambi´en medimos las tasas de germinacion,´ crecimiento de plantulas´ y de exogamia en pruebas de progenie. La variacion´ gen´etica de adultos no se correlaciono´ significativamente con el tamano˜ de la poblacion,´ como se esperaba, debido a que la fragmentacion´ ocurrio´ relativamente recientemente. Los efectos de la fragmentacion´ sobre la proge- nie no fueron severos. Solo´ los adultos presentaron endogamia significativa. Encontramos similar diversidad gen´etica total en los adultos y la progenie. Hubo baja diferenciacion´ gen´etica entre las poblaciones de adultos y de progenie. El crecimiento de plantulas´ se correlaciono´ positivamente con el numero´ efectivo de alelos, lo que muestra los efectos delet´ereos de la endogamia sobre la progenie. Las semillas provenientes de fragmen- tos pequenos˜ ten´ıan las mayores tasas de exogamia y de ´exito de germinacion,´ lo que indica que la mayor actividad de polinizadores en tales fragmentos redujo la autofecundacion´ , por lo tanto amortigua la erosion´ gen´etica y mantiene la variacion´ adaptativa. Los efectos de la fragmentacion´ del bosque fueron detectables en Embothrium coccineum, pero estos efectos probablemente no seran´ perjudiciales para la viabilidad de las poblaciones remanentes, porque las poblaciones fragmentadas mostraron altos niveles de flujo de genes y menor endogamia que las poblaciones de mayor tamano.˜ Las especies pioneras que no son sensibles a la tala de bosques pueden ser cruciales para los planes de recuperacion´ para facilitar el establecimiento de especies intolerantes a tales perturbaciones.

Palabras Clave: bosque templado, diversidad genetica,´ fragmentacion,´ tamano˜ poblacional, tasas de exogamia, vigor de la progenie

Introduction Temperate forests of austral South America are expe- riencing increasing deforestation (Armesto et al. 1998), Loss of continuous habitats and conversion to smaller with native forests of southern Chile having been nearly patches reduces population size rapidly and severely and exterminated during the second half of the twentieth cen- increases population isolation (Young et al. 1993). In par- tury (Aravena et al. 2002). Conversion of this region to ticular populations that suffer severe bottlenecks lose ge- mainly agricultural uses and plantations of exotic species netic variation because of founder effects, genetic drift, has affected the spatial continuity of several and an- and elevated inbreeding. Together these processes can imal species and the mutualistic interactions among them lead to fixation of deleterious alleles and inbreeding de- (Smith-Ram´ırez & Armesto 2003). pression, which can reduce viability and fecundity (Young We examined the impact of forest fragmentation on ge- et al. 1993, 1999; Couvet 2002). Isolated populations also netic traits of E. coccineum J.R. et G. Forster, a very com- tend to differentiate due to limited interpopulation gene mon tree in fragmented landscapes of southern Chile. An flow (Templeton et al. 1990; Ledig 1992). The result- analysis of satellite images for this region for 1976, 1985, ing decline in heterozygosis and allelic richness limit the and 1999 indicates that studied fragments are 20–25 years species’ ability to respond to changing selective regimes old, half of which were produced from one large ances- in the long term (Lesica & Allendorf 1995; Hamrick & tral population of approximately 130,000 ha (C. Echev- Nason 1996). err´ıa, personal communication). In addition the oldest Fragmentation and degradation of continuous forests and largest E. coccineum trees are found in patches of modify the habitat and thus affect the nature of plant– different sizes (Mathiasen 2004), suggesting that fragmen- animal interactions during different life-history stages, tation in southern Chile occurred relatively recently. such as pollination and seed dispersal (Aizen & Feinsinger We tested the hypothesis that trees in small fragments 1994). Nevertheless spatially isolated trees may facilitate and isolated trees are not genetically separated from the pollen movement (Nason & Hamrick 1997), connecting larger populations and thus experience normal or even fragments genetically (Guevara & Laborde 1993; Aldrich enhanced gene flow and do not suffer from inbreeding de- & Hamrick 1998). Conversely small populations may at- pression. We predicted that genetic diversity and progeny tract fewer pollinators, which may reduce pollen move- performance would vary directly with population size. A ment (Morgan 1999). Therefore the effects of fragmenta- limited effect of fragmentation on genetic structure of E. tion on plant species’ responses to pollinators depend on coccineum was expected because it is self-incompatible, compatibility systems and pollination mechanisms (Aizen so enforced outbreeding may counteract genetic erosion et al. 2002). In fragmented landscapes of southern Chile, and inbreeding in small fragments. We focused on forest pollinators visit individual Embothrium coccineum (Pro- fragments and spatially isolated trees of E.coccineum in teaceae) in isolation and in small forest fragments more a matrix of intense human use, which had been the sub- frequently than in medium and large fragments (Smith- ject of prior pollination studies (Smith-Ram´ırez & Armesto Ram´ırez & Armesto 2003). 2003). Because fragmentation occurred recently, we

Conservation Biology Volume 21, No. 1, February 2007 234 Genetic Effects of Fragmentation Mathiasen et al. consider adult trees representative of the genetic com- perimeter (measured with GIS satellite imagery) multi- position of forests before fragmentation. Therefore we plied by an approximately 2-m-wide band along fragment discuss our results in relation to population size, age class edges. Based on this approach, the six fragments included (adults vs. seedlings), time since fragmentation, depen- 47, 82, 97, 172, 934, and 990 individuals. These were dence on pollination vectors, and vigor of early progeny. identified as small fragments S-KS and S-ST, medium-sized fragments M-GB and M-WF,and large-sized fragments L-KC and L-KG, respectively.Isolated individuals were trees sep- Methods arated by at least 10 m and surrounded by pasture, identi- fied as I-MD and I-CP.Isolated trees were as old as trees in Study Species and Area forest fragments (Mathiasen 2004), indicating that these Embothrium is a monotypic genus in the , a remnants were part of the original continuous forest prior family of Gondwanic origin. It is endemic to temperate to fragmentation. We excluded isolated trees from statisti- forests of southern Argentina and Chile and ranges in the cal analysis of the effects of population size because they north from Linares (35◦ S) in Chile and Lago Norquinco˜ were widely dispersed across the landscape and could (39◦ S) in Argentina to Tierra del Fuego (55◦ S). Emboth- not be considered populations. rium coccineum grows as a tree or , usually associ- We measured germination and progeny vigor to assess ated with forest edges, although it never occurs as domi- whether inbreeding effects increased with population nant element or in pure stands. It is the only red-flowered size. We collected fruits produced by five inflorescences tree within austral forests. The bright red, tubular flow- from each of the canopies of 8–10 maternal trees of similar ers are hermaphroditic and open for 4 days, although the age in each fragment and from each isolated tree during dense inflorescences flower for up to 16 weeks (Smith- the austral summer of 2002. Each inflorescence produces Ram´ırez & Armesto 1994). Birds and a few insect species approximately five fruits, which resulted in a total sample visit E. coccineum flowers for nectar. On Chiloe´ Island, of 20–25 fruits per tree. Ten fruits from each maternal tree Chile, E. coccineum blooms during spring (September– were randomly selected to assess fruit mass, the number January) and is pollinated primarily by two bird species: of seeds per fruit, and average seed mass. The latter was Elaenia albiceps (flycatcher) and Sephanoides sephan- based on 10 batches of 10 randomly selected seeds from iodes (hummingbird: Smith-Ram´ırez & Armesto 2003). E. the total of the pooled fruits produced by a single mater- coccineum is self-incompatible in mountain areas at 41◦ S nal tree. Out of those total seeds produced per tree, we (Riveros 1991) and farther south at 50◦ S (Arroyo & Squeo randomly selected seeds that were maintained in progeny 1990), although it was recorded as self-compatible at low arrays to measure germination ability and progeny vigor. elevations at 39◦ S (Riveros et al. 1996). Seeds were germinated following Figueroa et al. (1996) in a greenhouse under natural light at Laboratorio Ecotono, We studied E. coccineum on northern Chiloe´ Island, ◦  ◦  between 41◦ 53–41◦ 59S and 73◦ 32–73◦ 41W. The Bariloche, Argentina (41 08 S, 71 19 W). study area consisted of forest fragments ranging from We determined germination success for 20 randomly 1 to more than 150 ha separated by a maximum of 15 selected seeds from each of the maternal families sampled km and isolated trees in a matrix of intense human land per site and counted the number of emerging seedlings use. Fragments in the study area were generated by forest every 7–10 days for 3 months. Seedling mortality was clearing during the past 100 years, and most were rem- recorded after 80 days as the proportion of germinated nants of continuous forests that covered Chiloe´ Island in seeds that died. Eight to fifteen of the remaining seedlings = the mid 1800s (Willson & Armesto 1996). Remnant veg- per maternal family (n 432) were grown in the com- etation was dominated by evergreen broadleaf species, mon garden for 3 months and then measured to quantify progeny vigor. We measured plant height, root length, including an emergent tree layer of endemic Myrtaceae, ◦ Drymis winteri J.R. et Foster (Winteraceae); two other and mass after drying seedlings at 40 C for 3 days. We Proteaceae avellana Mol. and hirsuta also used these seedlings, maintained in maternal fami- (Lam.) Diels ex J.F. Macbr.; southern beech (Nothofa- lies (i.e., progeny arrays), for genetic analysis of progeny gus nitida (Phil.) Krasser), and conifers such as Pilgero- and to analyze the relationship between vigor parameters dendron uviferum (D. Don) Florin (Cupressaceae) and with population and genetic traits. Podocarpus nubigena Lindl. (Podocarpaceae). Isozyme Electrophoresis Adult Sampling and Experiments on Seedling Performance We analyzed genetic characteristics of adult trees and We studied adult trees in six forest fragments and iso- their progeny from each fragment and the isolated trees. lated trees at two locations. The number of adult trees We considered that seedlings were representative of the in each fragment was estimated from the product of the current mating system interacting with the genetic struc- average density in four 25-m2 plots and the area occupied ture of the adult population(s). Fresh leaf tissue was by E.coccineum. This area was estimated as the fragment collected from approximately 50 adult trees per site and

Conservation Biology Volume 21, No. 1, February 2007 Mathiasen et al. Genetic Effects of Fragmentation 235 seedlings grown in a common garden. Adults included outcrossed paternity (rp) for progeny arrays from frag- the maternal trees from which we collected seeds and ments and isolated trees. We performed a hierarchical other randomly selected individuals. A total of 406 adult analysis of inbreeding by FIS as a measure of the aver- trees and 432 seedlings were analyzed by protein elec- age within-population inbreeding and FST as an indicator trophoresis with 12% starch gels (Starch Art Corporation, of among-population divergence, which—in case of a ge- Smithville, Texas). Nine enzymes coding for 22 putative netic equilibrium—is also an indirect estimator of gene isozyme loci were resolved in two buffer systems. We flow (Wright 1965, but see Whitlock & McCauley 1999). ran MDH (malate dehydrogenase, EC 1.1.1.37), PERc (ca- Finally we calculated total genetic diversity (Ht) for adult thodic peroxidase, EC 1.11.1.7), PGI (phosphoglucoiso- trees and seedlings based on polymorphic loci with FSTAT merase, EC 5.3.1.9), and SKDH (shikimate dehydroge- (version 2.9.3, Goudet 2002). nase, EC 1.1.1.25) in O’Malley et al.’s (1979) buffer sys- Several aspects of our analyses considered statistical tem. We used Poulik’s (1957) system to resolve IDH (isoc- hypotheses about means, which we tested with vari- itric dehydrogenase, EC 1.1.1.42), ME (malic enzyme, EC ous t tests. We used simple linear regression to inves- 1.1.1.40), MNR (menadione reductase, EC 1.6.99.2), PERa tigate the effects of log-transformed population size on (anodic peroxidase, EC 1.11.1.7), and PGM (phosphoglu- the genetic variation parameters determined on adult comutase, EC 5.4.22). Eleven of the 22 analyzed loci were trees and seedlings, germination rate, and mating-system polymorphic in at least one site, including MDH-2, MNR- parameters. We used stepwise multiple linear regres- 2, MNR-4, PERa-1, PERc-1, PERc-2, PERc-3, PGI-2, PGM-1, sion to analyze the relationships among average seedling PGM-2, and SKDH. vigor characteristics per family (plant height, root length, shoot weight, and total weight) and population charac- Genetic Parameters ters (seedling mortality, germination rate, seed weight, fruit weight, and number of seeds per fruit) and genetic We quantified genetic parameters separately for adult variation parameters (individual heterozygosity, polymor- trees and seedlings from each site because we considered phism, effective number of alleles, number of rare alleles, the two generations representative of pre- and postfrag- and mean number of alleles per locus). mentation mating environments. This analysis considered standard within-population parameters of genetic varia- tion and inbreeding estimated with POPGENE (version 1.32; Yeh et al. 1999), including mean number of alleles Results (A); effective number of alleles (A ); number of rare al- e Genetic Variation leles (Ar); percent of polymorphic loci under the sensu stricto criterion (Pss); observed heterozygosity (Ho); and Adult trees and seedlings from the six population frag- gene diversity (He). We estimated the number of rare al- ments differed somewhat in their genetic variation (Table leles following Premoli et al. (2000), as Ar = Alf [At/As], 1). Across the range of populations, seedlings were sig- where Alf is the number of low-frequency (<0.10) alleles nificantly more polymorphic (Pss) than adult trees (t7 = corrected by the number of alleles in a population (At) −2.77, p = 0.03, paired t test) and had fewer alleles (At) relative to the total number of alleles (As) observed in than adults (t7 = 3.58, p = 0.01, paired t test). Adults all populations. In addition we calculated the individual and progeny did not differ significantly for the other ge- heterozygosity for seedlings (Hi) as the number of het- netic parameters that we estimated ( p > 0.05 in all cases, erozygous loci to the total number of analyzed loci per paired t test). On average inbreeding coefficients within individual. adult populations were significantly different from zero We used the maximum-likelihood model of Ritland and (FIS ± SE = 0.324 ± 0.072; 95% confidence level [CL] = Jain (1981) and the MLTR (version 2.4, Ritland 2002) 0.171–0.545), whereas those of seedlings were not (0.095 computer program to estimate the following mating sys- ± 0.099; 95% CL =−0.045 to 0.535). Nevertheless both tem parameters: average single-locus (ts) and multilo- adults and progeny demonstrated a low, but significant, cus (tm) outcrossing rates, coefficient of biparental in- differentiation among populations (FST ± SE = 0.025 ± breeding (tm–ts: Brown 1990), and the correlation of 0.011; 95% CL = 0.006–0.059 and 0.055 ± 0.034; 95%

Table 1. Mean (SE) population estimates of genetic variation parameters for adult trees and seedlings of E. coccineum from six population fragments on Chilo´e Island, southern Chile.∗

n AAe Ar At Pss Ho He

Adults 50.8 (1.9) 1.66 (0.03) 1.40 (0.01) 3.26 (0.36) 37.1 (0.5) 42.6 (1.2) 0.13 (0.01) 0.196 (0.004) Seedlings 50.8 (7.7) 1.76 (0.04) 1.38 (0.01) 4.36 (0.68) 33.4 (0.8) 48.7 (1.6) 0.14 (0.01) 0.197 (0.004) ∗ Mean number of individuals sampled; A, mean number of alleles per locus; Ae, effective number of alleles; Ar, number of rare alleles; At, total number of alleles; Pss, polymorphism sensu stricto; Ho, observed heterozygosity; and He, gene diversity.

Conservation Biology Volume 21, No. 1, February 2007 236 Genetic Effects of Fragmentation Mathiasen et al.

lower multilocus outcrossing rates in one large fragment (tm = 0.78) and one location with isolated trees (tm = 0.53), suggesting that some selfing can occur. Multilocus and single-locus (ts) estimates of outcrossing were very similar (tm ± SE = 0.898 ± 0.060 and ts ± SE = 0.831 ± 0.039, respectively: t14 = 0.93, p = 0.37, t test) (Ta- ble 2), so the coefficient of biparental inbreeding (tm– ts) did not differ significantly from zero (t7 = 1.50, p = 0.18, unpaired t test), indicating little mating between relatives. The average correlation of outcrossed paternity (rp) among progeny within families was low (rp ± SE = 0.081 ± 0.028) but significantly greater than zero (t7 = 2.96, p = 0.02, unpaired t test), suggesting that randomly chosen pairs of outcrossed progeny from a maternal fam- ily tend to be full sibs (i.e., have the same pollen parent). Multilocus outcrossing rate (tm) decreased significantly Figure 1. Effects of log-transformed population size on with log-transformed population size (F1,4 = 13.02, p = gene diversity (He) of E. coccineum progeny in 0.02: Fig. 2), whereas the other mating-system parame- southern Chile. ters (ts, tm–ts, and rp) did not correlate significantly with this variable ( p > 0.1, linear regression). CL = 0.008–0.143, adults and progeny respectively). Fur- Progeny Performance thermore seedlings maintained similar higher overall total genetic diversity (Ht = 0.214) relative to adult trees (Ht = Germination rate was negatively correlated with log- 0.199) (t18 =−0.36, p = 0.76, paired t test by locus). transformed population size (F1,4 = 17.5, p = 0.01: Fig. 3). Genetic parameters correlated inconsistently with Seeds collected from intermediate fragments had higher population size. Genetic variability of adult trees and germination capacity than those from large fragments and seedlings from fragments did not correlate significantly isolated trees (Fig. 3), which in turn showed comparable with log-transformed population size ( p > 0.1 in all cases, germination rates (percent germination attained was 35.6 simple linear regressions), although seedlings showed a [L-KC] and 20.0 [L-KG] in large fragments and 34.8 [I-MD] tendency to exhibit higher gene diversity (He) in large and 25.0 [I-CP] in isolated trees). fragments (Fig. 1). As expected isolated adult trees exhib- Average measures of seedling size after 3 months did ited similar genetic variation to those in forest fragments not correlate significantly with log-transformed popula- ( p > 0.05 in all cases, t tests). However seedlings pro- tion size ( p > 0.1 in all cases, simple linear regression) duced by isolated trees had significantly fewer rare alleles but were significantly related to the effective number of (Ar = 2.21) (t1 =−215, p = 0.003, unpaired t test) and alleles and, in some cases, seedling mortality (Table 3). lower observed heterozygosity (Ho = 0.13) (t1 =−21, Seedlings from families with a large effective number of p = 0.03, unpaired t test) than those produced by adults alleles were taller, had longer roots and weighed more from forest fragments (Ar = 4.36 and Ho = 0.14). than those with fewer alleles. In addition the surviving Embothrium coccineum primarily outcrosses on seedlings from families with poor seedling survival tended Chiloe´ Island (multilocus outcrossing rates (tm) in most to be shorter and lighter than those from families with locations exceeded 0.9, Table 2). We detected somewhat high survival.

Table 2. Population size and mean (SE) estimates of mating system parameters for isolated E. coccineum (sites I-MD and I-CP) and six population fragments in Chiloe´ Island, southern Chile.∗

Site Size tm ts tm-ts rp

I-MD NA 0.534 (0.272) 0.711 (0.176) −0.177 (0.199) 0.240 (0.399) I-CP NA 0.921 (0.262) 0.917 (0.218) 0.004 (0.139) 0.126 (0.137) S-KS 47 1.063 (0.121) 0.963 (0.067) 0.100 (0.131) 0.008 (0.071) S-ST 82 0.983 (0.132) 0.710 (0.093) 0.273 (0.082) 0.053 (0.068) M-GB 172 1.007 (0.240) 0.945 (0.149) 0.063 (0.137) 0.008 (0.031) M-WF 97 0.993 (0.190) 0.884 (0.153) 0.109 (0.119) 0.073 (0.135) L-KC 990 0.905 (0.099) 0.806 (0.048) 0.099 (0.080) 0.111 (0.218) L-KG 934 0.775 (0.377) 0.714 (0.160) 0.061 (0.335) 0.031 (0.024) ∗Abbreviations: tm, multilocus outcrossing rate; ts, single-locus outcrossing rate; tm–ts, biparental inbreeding coefficient; and rp, correlation of outcrossed paternity; NA, not applicable.

Conservation Biology Volume 21, No. 1, February 2007 Mathiasen et al. Genetic Effects of Fragmentation 237

Discussion

Fragmentation is probably affecting the genetic variability of E. coccineum populations in southern Chile. Reduced allelic richness in progeny, as measured by the total num- ber of alleles and the tendency for a higher gene diver- sity of progeny with population size, most likely reflects local mate availability and mating relationships within and among fragments. Whether these have changed as a consequence of fragmentation is still a bit uncertain. Genetic theory predicts that small and isolated popula- tions have lowered genetic variation (Nei et al. 1975), as documented in remnant habitats of other species con- taining few reproductive individuals after fragmentation (Young et al. 1999; Tomimatsu & Ohara 2003). In con- trast to shade-tolerant species that depend on facilitation Figure 2. Effects of log-transformed population size on mechanisms including the availability of secure sites for multilocus outcrossing rate (tm) for forest fragments establishment, early colonizers such as E. coccineum vig- of E. coccineum in southern Chile. orously regenerated in cleared areas and therefore popu- lation size may even increase under fragmentation. Hence the genetic and demographic consequences of fragmen- tation are closely linked to autoecological traits. Isozyme variation of adults was moderate and not re- lated to population size. We speculate that the genetic variation of adult E. coccineum trees may reflect a struc- ture that was probably established prior to habitat frag- mentation. Given that fragmentation in the study area occurred recently (C. Echeverr´ıa, personal communica- tion), too little time has elapsed for fragmentation to pro- duce noticeable genetic effects on adults and only weak effects on progeny. In addition current genetic structure may be a reflection of the fact that the species occurred naturally in small patches within the closed canopy and was rarely found as continuous forest. Comparable results have been found in several other species. In Vincetoxicum hirundinaria, a perennial herb, genetic characteristics are not directly related to Figure 3. Relationship of the germination rate of E. population size but rather to the demographic history and coccineum seeds from forest fragments in Chilo´e mating system of the species (Leimu & Mutikainen 2005). Island, Chile, to log-transformed population size. Similarly genetic diversity did not vary with current popu- lation size in long-lived iaspicula and G. caleyi (Proteaceae), which had suffered recent fragmentation

Table 3. Summary statistics for stepwise multiple regression analyses ( p < 0.05) of seedling size after 3 months against life-history characters and genetic traits (independent variables) of E. coccineum.

2 a 2 Dependent variables R F df p Independent variables β R partial p

b Plant height 0.295 2.72 4,26 0.05 Ae 0.589 0.176 0.02 b b Root length 0.343 3.39 4,26 0.02 Ae 0.799 0.132 0.01 Shoot weight 0.477 4.03 5,25 0.01b M −0.430 0.184 0.01b Ae 0.359 0.101 0.03 Total weight 0.327 4.36 3,27 0.01b M −0.370 0.160 0.03 Ae 0.327 0.098 0.05 a Key: Ae, effective number of alleles; M, percentage of seedling mortality after 80 days. bSignificant after Bonferroni correction (Rice 1989).

Conservation Biology Volume 21, No. 1, February 2007 238 Genetic Effects of Fragmentation Mathiasen et al.

(Hoebee & Young 2001; Llorens et al. 2004). In these However selfing may occur, as indicated by relatively low species self-incompatibility and the production of seed outcrossing rates in one large fragment (tm = 0.775) banks may buffer the loss of diversity, even following se- and one location with isolated trees (tm = 0.53). The vere reductions in population size (Levin 1990). In con- large fragment (L-KG) was the only study site immersed trast predominantly outcrossed, self-compatible species in a matrix dominated by Baccharis (Asteraceae), an may be more susceptible to the genetic effects of fragmen- insect-pollinated shrub (Aizen & Ezcurra 1998). The iso- tation. This is due to higher inbreeding in small popula- lated trees at site I-MD occurred on private property tions and the expression of lethal recessive alleles when with beehives, where bees visit E. coccineum frequently homozygosity increases (Charlesworth & Charlesworth (C. Smith-Ram´ırez, personal communication). Although 1987). open-pollinated families have paternity with low average Similar genetic diversity in adults and progeny is main- correlation (rp < 0.13), isolated trees at I-MD had the high- tained by the predominant self-incompatibility of E. coc- est (rp = 0.24). Among these isolated trees, it is likely that cineum, which creates a dependence on pollinators to drift has led to fewer compatible mates and only a few fa- facilitate mating. Self-incompatibility may favor pollen im- thers contributed to each seedling family, which resulted ported from other fragments, particularly where few re- in a higher rp. Hence in addition to fragment size, the productive trees are available for successful pollination. mating system and the genetic structure of E. coccineum Previous data revealed that the main pollinator of E. coc- were affected by pollinator assemblages and the vegeta- cineum in the study area, the bird E. albiceps (Tyrannidae, tion associated with the surrounding matrix. Passeriformes), frequently visits spatially isolated individ- Seed germination was negatively correlated with pop- uals and those at forest edges within small fragments ulation size (Fig. 3), which may reflect higher inbreeding (Smith-Ram´ırez & Armesto 2003). In contrast pollinators in large fragments. Deleterious effects of inbreeding are are more territorial in larger fragments, where they tend also evident in the positive relationship of seedling per- to defend three to five adjacent flowering trees on which formance with effective number of alleles (Table 3). Such they forage for nectar. Moreover in such large fragments, inbreeding presumably results from selfing and/or mating nearby E. coccineum individuals tend to be genetically between relatives caused by short interplant movements similar because seeds primarily disperse within 20 m of of pollinators as measured in large fragments. Open areas the maternal tree (Rovere & Premoli 2005). Hence trees in were characterized by abundant regeneration of E. coc- larger fragments may commonly inbreed if pollinator be- cineum from only few reproductive individuals, which havior favors mating between related neighboring may pose demographic problems. The loss of adaptive in such structured populations. This biparental inbreed- genetic variation and inbreeding depression may threaten ing may explain the lower percentage of fruit production population viability in this fragmented landscape (Ooster- in medium-sized and large fragments (5% and 6%, respec- meijer et al. 1995; Reed & Frankham 2003) because they tively) compared with that in small fragments and isolated may alter vital characteristics of the population (Sherwin trees (11% and 13%, respectively) within the study area & Mortiz 2000) and reduce the population’s ability to (A.E.R., unpublished data). In addition, larger fragments respond to selective pressures posed by the changing en- are probably composed of several small demes that may vironment (Reed & Frankham 2003). suffer higher drift than isolated trees and individuals at To maintain genetic diversity and fitness, forest patches forest margins and in small fragments, where genes are of pollinator-dependant species such as E. coccineum more freely exchanged (Aldrich & Hamrick 1998). must be preserved. These fragments will balance genetic Low genetic divergence among adults (FST = 0.025) erosion by drift, reduce inbreeding, and retain sufficient and seedlings (FST = 0.055) suggests that there is little heterozygosity through a continuous influx of alleles from restriction of gene flow (Nm > 1) among populations locally distinct gene pools mediated by pollinators. Iso- inhabiting fragmented landscapes. Nevertheless, within- lated trees may act as connectors, maintaining gene flow population inbreeding seems to follow distinct patterns among distant populations (Aldrich & Hamrick 1998; Cas- in adults and progeny. On one hand adults exhibit higher cante et al. 2002; White et al. 2002). Our results indicate mean inbreeding (0.324) than seedlings (0.095), perhaps that spatially isolated trees and forest remnants are suf- reflecting prefragmentation Wahlund effects and thus the ficient for the maintenance of viable populations under- existence of a former population structure (Young et al. going fragmentation. Embothrium coccineum can then 1996). In contrast reduced inbreeding of progeny cor- be considered to have low sensitivity to fragmentation, relates with the observed high outcrossing rates. Hand- which mitigates the effects of genetic erosion. This to- pollination experiments in the study area indicate that gether with its natural ability to colonize open areas facil- outcross and open pollination yielded significantly higher itate the establishment of other shade-tolerant plants that fruit set (37.3% and 17.3%, respectively) than sponta- in turn are highly sensitive to fragmentation. neous autogamy or self-pollination (0% and 1.3%, re- Fragments are all that remain of native forests in south- spectively; Rovere et al. 2006). These results are con- ern Chile because of intense land use, including plan- sistent with a predominantly outcrossing mating system. tations of exotic species. We studied an area within

Conservation Biology Volume 21, No. 1, February 2007 Mathiasen et al. Genetic Effects of Fragmentation 239 administrative Region X, which supports 7.5% of Chile’s Brown, A. H. D. 1990. Genetic characterization of plant mating systems. remaining lowland and coastal mountain forests (Neira et Pages 145–162 in A. H. D. Brown, M. T. Clegg, A. L. Kahler, and B. al. 2002). These forests are in turn the least represented in S. Weir, editors. Plant population genetics, breeding, and genetic resources. Sinauer Associates, Sunderland, Massachusetts. the protected-area system. Austral forests of South Amer- Cascante, A., M. Quesada, J. J. Lobo, and E. A. Fuchs. 2002. Effects of ica have an endemic biota at high taxonomic levels. Their dry tropical forest fragmentation on the reproductive success and flora also exhibits one of the highest incidences of bi- genetic structure of the tree Samanea saman. Conservation Biology otic pollination (23%) of woody plants, particularly or- 16:137–147. nithophily, which is comparable to incidences in tropical Charlesworth, D., and B. Charlesworth. 1987. Inbreeding depression and its evolutionary consequences. Annual Review of Ecology and areas (Aizen et al. 2002). Nevertheless, in contrast to the Systematics 18:237–268. tropics, these ornitophilous plants depend strongly on a Couvet, D. 2002. Deleterious effects of restricted gene flow in frag- few bird species. For example, approximately 20% of red- mented populations. Conservation Biology 16:369–376. flowered woody plants are visited by one hummingbird Figueroa, J., J. J. Armesto, and J. F. Fern´andez. 1996. Estrategias de ger- species (S. sephaniodes; Smith-Ram´ırez 1993). Areas in minacion´ y latencia de semillas en especies del bosque templado de Chiloe,´ Chile. Revista Chilena de Historia Natural 69:243–251. temperate latitudes with elevated endemism and highly Goudet, J. 2002. FSTAT, a program to estimate and test gene diversi- dependant mutualistic interactions could be as fragile as ties and fixation indices (version 2.9.3.2). Universite´ de Lausanne, those in the tropics, so conservation practices should em- Dorigny, Switzerland. Available from http://www2.unil.ch/popgen/ phasize the role of remnant forest fragments. softwares/fstat.htm (accessed February 2002). Guevara, S., and J. Laborde. 1993. Monitoring seed dispersal at desolated standing trees in tropical pastures: consequences for local species availability. Vegetatio 107/108:319–338. Acknowledgments Hamrick, J. L., and J. D. Nason. 1996. Consequences of dispersal in plants. Pages 203–236 in O. E. J. Rhodes, R. K. Chesser, and M. H. We thank personnel at Biological Station Senda Darwin for Smith, editors. Population dynamics in ecological space and time. University of Chicago Press, Chicago. logistic support during fieldwork and C. Smith-Ram´ırez Hoebee, S. E., and A. G. Young. 2001. Low neighbourhood size and high and J. Armesto for early discussions on the development interpopulation differentiation in the endangered shrub Grevillea of the research plan. M. Arbetman, N. Brito, P. Quiroga, iaspicula McGill (Proteaceae). Heredity 86:489–496. and C. Souto provided valuable assistance in the labo- Ledig, F. T. 1992. Human impacts on genetic diversity in forest ecosys- ratory. We are most grateful to L. Harder, G. Oostermei- tems. Oikos 63:87–108. Leimu, R., and P. Mutikainen. 2005. Population history, mating system, jer, and P. Aldrich for their valuable comments on our and fitness variation in a perennial herb with a fragmented distribu- manuscript. This research was funded by the European tion. Conservation Biology 19:349–356. Commission under Framework IV of DGXII as part of Lesica, P., and F. W. Allendorf. 1995. When are peripheral populations the BIOCORES project (PL ICA4-2000-10029). P.M. and valuable for conservation? Conservation Biology 9:753–760. 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