The Biogeography of the Caribbean Land Snail Family Annulariidae

Home , Abbottella, Annulariidae, Chondropoma, Girardinus, Littorina brevicula, Littorinoidea

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Nicholas David Skomrock, B.S.

Graduate Program in Evolution, Ecology and Organismal Biology

The Ohio State University

2014

Thesis Committee:

Meg Daly, Advisor

John Freudenstein

G. Thomas Watters

Nicholas David Skomrock

2014

Abstract

The complex nature of historical Caribbean geology confounds the understanding of biogeographic information and the reconstruction of historical biogeographic patterns.

Explanations of distributions of terrestrial organisms have included over-water disperal, land dispersal and vicariance, with two main explanations being the vicariance model of

Rosen (1975) and the GAARlandia hypothesis of Iturralde-Vinent and MacPhee (1999).

The land snail family Annulariidae, endemic to the Caribbean, has shown similar patterns to other organisms in the region and is used to help evaluate the relative role of over water dispersal, land dispersal and vicariance. A molecular phylogeny using regions of the nuclear ribosomal 28S, mitochondrial ribosomal 16S, and the mitochondrial protein coding gene COI was reconstructed and used to assess patterns of biogeography.

Ancestral ranges were reconstructed using the program RASP with the BBM and S-diva algorithms. These analyses suggest a proto-Antillean origin of the fauna with subsequent over-water dispersals, relying heavily on land or over-water dispersals as a mechanism.

Little evidence was found in support of the GAARlandia hypothesis, yet it cannot be completely removed as a potential explanation. Herein, we describe the historical biogeographic patterns of the present day Annulariidae.

Acknowledgments

I would like to thank the following for help providing specimens: Paulo Albano, Kurt

Auffenberg, Kevin Cummings, Glenn Duffy, Zoltan Fehér, Alejandro Fernández

Velázquez, Raúl Fernández-Garcés, Steffen Frank, Alan Gettleman, Rich Goldberg, Jozef

Grego, Adrienne Jochum, Homer Rhode, Emilio Rolán-Alvarez, John Slapcinsky, Jozef

Šteffek, Fred Thompson, and G. Thomas Watters. I would also like to thank Meg Daly,

John Freudenstein, Kelsey Fultz, Paul Larson, Jason Macrander, Abby Reft and Brandon

Sinn for assistance in the lab.

iii

Vita

June 2006 ...... Harvey High School

June 2010 ...... B.S. Evolution and Ecology, The Ohio State

University

2012 to 2013...... Metro Fellow, The Ohio State University

2011 to present ...... Graduate Teaching Associate, Department

of Evolution, Ecology and Organismal

Biology, The Ohio State University

Fields of Study

Major Fields: Evolution, Ecology and Organismal Biology

Statistics

Table of Contents

Abstract...... ii

Acknowledgments...... iii

Vita...... iv

Table of Contents...... v

List of Figures...... vii

List of Tables ...... viii

Introduction...... 1

Materials and Methods...... 5

Taxon and gene sampling ...... 5

Phylogenetic analyses ...... 7

Ancestral Range Reconstruction...... 8

Results...... 10

Taxon and gene sampling ...... 10

Phylogenetic analyses ...... 15

Ancestral Area Reconstruction ...... 20

v Discussion...... 22

Origins...... 22

Biogeographic Patterns ...... 23

Temporal relationships...... 25

Evidence for dispersal...... 26

Challenges and future directions...... 27

Conclusion ...... 28

References...... 29

List of Figures

Figure 1 Species richness and sampling coverage...... 6 Figure 2 Maximum likelihood reconstruction of the full data set ...... 17 Figure 3 BBM Analysis on subset data set ...... 19 Figure 4 Ancestral range reconstruction using S-Diva...... 21

vii

List of Tables

Table 1 List of taxa represented in this study...... 10

viii

Introduction

The Caribbean is a geologically complex region whose incompletely-known history confounds the interpretation of biogeographic and distributional data for the species found there (Hedges 2001, Crews and Gillespie 2010). The distributions of terrestrial organisms within this region have been explained with reference to three primary models: overwater dispersal, land dispersal and vicariance. These have been inferred to reflect possible geologic patterns but have long been debated (Hedges 2001).

Historically, biogeographers described patterns of island biogeography as the result of overwater dispersal from one landmass to the other, which was supported by the subsequent decrease in diversity from island to island. Starting at the mainland, organisms transferred to the closest islands and “hopped” across to islands further away through mechanisms of wind or water. Several current studies invoke this as a primary or secondary mode to explain distributions across different taxa (e.g. Hedges 2006,

Michelangeli et al. 2008). This concept was the primary explanation in the Caribbean until the idea of vicariance was applied to this region by Rosen (1975).

The vicariance model of Rosen (1975) posits that there were islands that linked

Central and South America, called the proto-Antilles, through which organisms were continuously distributed. The proto-Antilles separated via the subduction of the

Caribbean Plate between the North and South American plates, fanning out into the

1 present-day Antilles and leading to vicariant speciation in the lineages endemic to the islands. This model predicts the origins of the terrestrial organisms in the Greater and

Lesser Antilles to be Central America with radiations to the proto-Antilles, which later diverged concordantly with the separation of land masses.

An alternative terrestrial route for dispersal was proposed as the GAARlandia hypothesis by Iturralde-Vinent and MacPhee (1999). The GAARlandia hypothesis proposes a recent land bridge throughout the Caribbean that facilitated the movement of land organisms from South America through the Lesser Antilles via the Aves Ridge and into the Greater Antilles with subsequent vicariance (Iturralde-Vinent and MacPhee

1999). This hypothesis suggests Caribbean fauna originated in South America, subsequently invading the Greater Antilles through the landbridge composed of the Aves

Ridge.

Both the vicariance and GAARlandia models hypothesize landmasses whose presence or availability for colonization have not been confirmed from geological evidence (Hedges 2001). The geologic models describing the origin of the Carribean are not always consistent, but the model with what appears to have the most support is that of

Pindell and Barrett (1990) (reviewed in Graham 2003). This model describes the emergence of the Antilles as already formed submerged islands that were uplifted in a sequential pattern starting in Cuba and moving eastward across the Greater Antilles and through the Windward Islands. This suggests that the proto-Antilles were never a continuous chain of islands, conflicting, in part, to the vicariance model since vicariance requires a new physical separation. Because of this, explanations of the biogeography of

Caribbean-endemic terrestrial organisms must include overwater dispersal between 2 islands, which has been argued by other workers (Graham 2003, Hedges 1996). Pindell and Barrett’s (1990) model also does exclude the possibility of GAARlandia, but does not discuss the availability of the Aves Ridge as a landmass for land dispersal.

The vicariance and GAARlandia hypotheses also differ in the timing of events.

The proto-Antilles are believed to have initially separated during the late Cretaceous with colonization beginning shortly after the K-Pg boundary (Rosen 1975). The GAARlandia land bridge was hypothesized based on geologic evidence to have been present for about

3 million years around 35-33 MYA (Iturralde-Vinent and MacPhee 1999). Molecular dating techniques have been used to help assess the timing of divergences in order to show support for the GAARlandia hypothesis. These studies dated their phylogenies and evaluated the separation of lineages to the proposed availability of GAARlandia (e.g. in

Crotons, Van Ee et al 2008; in butterflies Wahlberg 2006; in toads, Alonso et al. 2012).

However, several studies implementing a molecular clock find divergence times within lineages that conflict with the expectations of the GAARlandia hypothesis suggesting that the patterns of diversification are inconsistent with the expectation under this hypothesis

(e.g. in Squamates, Giugliana 2008; in Girardinini, Doadrio et al. 2009).

The species rich land snail family Annulariidae (Gastropoda: Littorinoidea) can provide a unique perspective on Caribbean biogeography because its 700 members are terrestrial but have the capability for easy overwater dispersion (Watters 2006). These snails can use the tight-fitting operculum to seal themselves in their shell for long periods of time, thus enabling them to withstand variability in their environment (Rees 1964,

Watters 2014b). Some Annulariids also possess breathing devices to allow them to obtain oxygen while sealed within their shells, potentially increasing the time permitted for 3 dispersal. Living primarily on rocky outcrops, in caves and in forested areas, this group of snails generally has species that are endemic to small areas – some may inhabit a single cliff side or cave (Watters 2006).

The perspective on Caribean biogeography afforded by study of the Annulariidae is especially critical because many previous assessments of Caribbean biogeography have focused on vertebrates, which may have different constraints on their dispersal (eg.

Hedges et al. 1996, Davalos 2004) than these small-bodied, slow-moving animals. As is true of many other organisms within the region, members of this family are extreme calciphiles, highly endemic and species often only appear in small areas (Watters 2006), which is necessary for a clear, cladistic analysis of the biogeographical patterns (e.g. Page and Lydeard 1994, Morrone and Crisci 1995).

There has been no previous cladistic biogeographic assessment of this group, but there have been several descriptive texts that address biogeographic patterns. The biogeographic pattern expected for these snails suggests a joint vicariance and dispersal model with origins in Central America (Watters 2006, Watters 2014a,b). Here I test those expectations by constructing a phylogenetic framework for Annulariidae, and use this phylogeny to evaluate the utility of invoking GAARlandia as a plausible explanation for the distribution of these taxa. By using a molecular, phylogenetic approach with model based assessments, I describe the biogeographic patterns in relation to models of geologic formations.

Materials and Methods

Taxon and gene sampling

Sampling of the Annulariidae was based primarily on geography, and secondarily on the current taxonomy of the group following Watters (2006), but under the constraint of availability since collecting was not possible for many regions (e.g. western Cuba.

Samples that were not obtained through recent collections were borrowed from museums

(Figure 1, Table 1). Outgroups were selected from the available members of the

Pomatiidae, the presumed closest relative of the Annulariidae, and sequences available from Genbank of Littorina brevicula, representing Littorinidae, the other family within

Littorinoidea.

Figure 1 Relative species richness of the Annulariidae (Top) and sampling coverage of this study indicated in red (Bottom) (Modified from Watters)

6 DNA was extracted from a portion of the foot in larger specimens or the entire animal if it was small using the Qiagen DNeasy Animal Tissue Spin Column protocol.

For specimens from museum collections or those with unknown preservation history, the extraction protocol was modified as follows: whole specimens were flash-frozen in liquid nitrogen and crushed with a pestle prior to tissue lysis.

DNA sequences were collected for three markers: a region of the nuclear ribosomal 28S, mitochondrial ribosomal 16S, and the mitochondrial protein coding gene

COI, all of which have been used successfully in other studies of molluscan genera and families (e.g. Reid, Rumbak and Thomas 1996, Park and O’Foighil 2000). A portion of the nuclear ribosomal 28S gene, spanning regions D1-D6, was amplified with the primer combinations of either D1f-D4Rb and D23f-D6r, or D1f-D24r, D23f-D4Rb and D4f-D6r using the cycling parameters described in Park and O’Foighil (2000). 16S was amplified with primers 16Sar and 16Sbr as described in Reid, Rumbak and Thomas (1996). COI was amplified with the LCO1490 and COR722b and cycling parameters of Smolen and

Falniowski (2008). Internal primers were developed for COI to assist in amplification of degraded DNA using sequences generated in this study.

Sequences were assembled in Sequencher (Gene Codes 2000) and queried against the nucleotide database of GenBank using BLAST (Altschul et al. 1997) to verify that sequences were from a gastropod; any sequences that were returned as most similar to non-gastropod organisms were removed from the study.

Phylogenetic analyses

7 Two data sets were created since many biogeography programs have constraints on the number of taxa and the number of localities permitted as input. The full data set consisted of all sequences sampled and used Littorina brevicula as the outgroup from the family Littorinidae, the presumed sister group to the Annulariidae and the Pomatiidae

(Watters 2006). The taxa for the subset analysis were selected for completeness of the sequences available, attempting to cover the greatest diversity of geographic ranges and genera, requiring taxa selected to have at least the markers COI and 28S and preference given to those with all three markers. The subset taxa were then selected for genera available and all major land masses, attempting to have at least one individual from each.

Pomatias elegans, from the family Pomatiidae was selected as the outgroup for the subset analysis.

PartitionFinder (Lanfear et al. 2012) was used on both data sets to determine the best substitution and partition models for RaXML and MrBayes inputting the potential partitions as the codon position in COI, plus the entire markers of 16s and 28s, for a possible maximum of five partitions. Phylogenetic reconstructions were performed in

RaXML (Stamatakis 2006), MrBayes (Ronquist et al. 2012), and TNT (Goloboff et al.

2008) on both the subset and full data sets using the partitions and models found. RaXML and MrBayes were ran on Cipres Portal (Miller et al. 2010).

Ancestral range reconstruction

Since ancestral reconstructions implementing a Dispersal-Extinction-

Cladogenesis (DEC) model often require ultrametric trees or the fitting of a molecular clock, the assumption of a relaxed molecular clock was assessed in MrBayes using Bayes

8 factors of the approximate likelihood scores on the subset data. The standard model was fit to the data based upon the previous analyses and the stepping stone procedure was used to assess an accurate estimate of the likelihood. The molecular clock model was independently fit to the data using a uniform prior of node times and a clock rate prior

TK02 (lognormal relaxed model) as described in Lepage et al. (2007) who found these to be these to be the best fit when lacking significant prior information. The standard model was accepted as the better fit model, having a Bayes factor of about 140. This lack of support for a molecular clock limited subsequent analyses.

Ancestral range reconstruction was performed on the subset data in the program

RASP (Yu et al. 2011) using Bayesian Binary MCMC (BBM) and S-Diva analyses. The input trees for both analyses were from the sampling of the posterior distribution outputted from MrBayes after excluding the burn-in. Each terminal was assigned a geographic locality based upon collection location, assuming the species was found only at a single location since there has been no evidence to the contrary of this assumption.

The possible geographic localities were: Bahamas, Cuba, Hispaniola excluding the

Tiburon Peninsula, Tiburon Peninsula, Jamaica, Upper Lesser Antilles (which includes

Puerto Rico and the Virgin Islands), Lower Lesser Antilles (ABC islands), Caymans, and

Central America. Specimens yielding confident sequences for all of the markers were not obtained from the Yucatan Peninsula, so the area was not included in the analysis.

Analyses were implemented using the default settings.

Results

Taxon and gene sampling

The 103 in-group specimens for which I generated new data represent 91 species and 37 genera. The full data set concatenated alignment contains a maximum of 2442 nucleotides and has 28.7% of the sequence data missing. The subset alignment has 37 in- group taxa, a maximum concatenated alignment length of 2356 aligned nucleotides and

3.8% missing sequence data. The geographic sampling for both the full and subset taxa cover the region and have multiple representatives from the most diverse areas. Figure 1 shows the number of species described (top) and the sampling present in the full data set

(bottom). Taxa represented in this study can be found in Table 1.

Table 1 List of taxa represented in this study.

Taxon Source Locality 28s 16s COI Abbottella milleacantha Coll. J. Guaraguao, Dominican X Watters & Duffy, 2010 Grego Republic Abbottella urbana UF 249110 San Cristobal, X X Watters, 2012 Dominican Republic Adamsiella ignilabris (2) Jamaica X X X Adamsiella ignilabris (3) Jamaica X X X Adamsiella ignilabris UF 249132 Norwood, Jamaica X (Adams, 1849) Adamsiella irrorata Jamaica X X X Adamsiella variabilis (3) Jamaica X X X Cont.

10 Table 1 Cont. Adamsiella variabilis UF 249120 Prickly Pole, Jamaica X X X (Adams, 1849) (1) Adamsiella variabilis UF 249115 Christiana, Jamaica X X (Adams, 1849) (2) Aguayotudora bermudezi UF 119104 Sibanucú, Cuba X (Torre & Bartsch, 1941) (1) Annularia chittyi UF 249136 Jacob Hut, Jamaica X X X (Adams, 1849) Annularia fimbriatula Jamaica X X X Annularia lima Jamaica X X X Annularia lima (Adams, UF 249124 Planters Hall, Jamaica X 1845) Annularia pisum UF 249122 Ecclesdown, Jamaica X X (Adams, 1849) Annularia retrorsum Jamaica X X X Annularia sp. Jamaica X X X Annularia spinulosa Jamaica X X X Annularisca roemeri GTW 15037a Yumurí, Cuba X X X (Pfeiffer, 1864) Annularisca victoris GTW 7163e Playa El Baga, Cuba X X X (Torre & Bartsch, 1941) Articulipoma woodringi UF 249173 Hato Major, Haiti X X X (Bartsch, 1946) Chittypoma monstrosum Jamaica X X X Choanopomops UF 207138 Neuendorf, Belize X X X largillierti (Pfeiffer, 1846) (1) Chondropoma blauneri UF 249160 Cerro de las Cuevas, X X (Shuttleworth, 1864) Puerto Rico Chondropoma laetum GTW 15048a Velasco, Cuba X X X appendiculatum Torre & Bartsch, 1938 Chondropoma pictum UF 119466 Matanzas, Cuba X Pfeiffer, 1839 Chondropoma pupiforme UF 48724 Anguilla X X (Sowerby, 1843) Chondropoma solidulum GTW 15056a Playa Morales, Cuba X X X melanaxis Aguayo, 1943 Chondropoma sp. 1 GTW 15046a Guardalavaca, Cuba X X X Chondropoma sp. 3 GTW 15055a Banes, Cuba X X X Chondropoma sp. 4 GTW 15050a Rafael Freyre, Cuba X X Cont. 11 Table 1 Cont. Chondropomella elegans Dominican Republic X Chondropomium GTW 7170d Pedernales, Dominican X X X marmoreum (Watters & Republic Duffy, 2010) Chondropomium sp. UF 78234 Robin, Haiti X X Chondropomium UF 249185 Boca de Cachón, X superbum (Henderson & Dominican Republic Simpson, 1902) Chondropomium swifti UF 33092 Anse-Rouge, Haiti X X (Shuttleworth, 1854) Chondropomorus sp. UF 48103 Peake le Selle, Haiti X X Chondrothyrium GTW 15047a Topes de Collantes, X violaceum (Pfeiffer, Cuba 1852) Cistulops raveni (Crosse, UF 217791 Tofelberg, Curaçao X X X 1872) Clydonopoma alyshae UF 249196 Azua, Dominican X X (Watters & Duffy, 2010) Republic (1) Clydonopoma nobile GTW 7089b Virgen de San Rafael, X X X (Pfeiffer, 1852) Dominican Republic Clydonopoma pumilum GTW 7172b Las Mercedes, X X X (Watters & Duffy, 2010) Dominican Republic Colobostylus chevalieri UF 249228 Johnson Hall, Jamaica X X (Adams, 1851) Colobostylus Jamaica X X X humphreysianus Colobostylus jayanus (2) Jamaica X X X Crossepoma GTW 7088b Oviedo, Dominican X X X vermiculatum (Bartsch, Republic 1946) Cubadamsiella beneitoi GTW 15257a Cumanayagua, Cuba X Fernández-Garcés, Espinosa & Ortea, 2004 Diplopoma aquadillense OSUM 4168 Mona Island X X turnerae (Clench, 1950) Diplopoma crenulatum UF 259784 Grand Fond, X (Pfeiffer, 1839) (1) Guadeloupe Diplopoma pujalsi GTW 15045a Mayarí, Cuba X X X (Aguayo, 1953) Diplopoma rigidulum UF 146287 Benque Viejo del X X (Morelet, 1851) Carmen, Belize Cont. 12 Table 1 Cont. Diplopoma storchi GTW 15049a Cantera de Mella, Cuba X X X (Pfeiffer, 1962) Eyerdamia bertini UF 125256 Elias Piñas, Dominican X (Maltzan, 1888) Republic Gouldipoma chrysostiria UF 221352 Cerro Piedra Blanca, X X X Watters, MS Honduras Gouldipoma plicatulum UF 246535 San Esteben, X (Pfeiffer, 1846) Venezuela Gouldipoma trochlea UF 159408 Agua Blanca, Mexico X (Pfeiffer, 1852) Halotudora aripensis UF 77048 Arima, Trinidad X (Guppy, 1864) (1) Halotudora gruneri UF 146289 Benque Viejo del X X (Pfeiffer, 1846) (1) Carmen, Belize Halotudora kuesteri UF 207684 San Felipe, Belize X (Pfeiffer, 1852) Lagopoma lagopoma UF 218070 El Valle, Dominican X X Bartsch, 1946 (1) Republic Meganiphe rhecta UF 249213 Yaroa, Dominican X X Thompson, 1978 Republic Opisthosiphon UF 247198 Long Island, Bahamas X X X bahamasense (Pfeiffer, 1865) (1) Opisthosiphon OSUM 6344 Andros Island, X X bahamasense (Pfeiffer, Bahamas 1865) (2) Opisthosiphon coloni UF 246650 San Salvador Island, X Bartsch, 1946 Bahamas Opisthosiphon GTW 8708c Eleuthera, Bahamas X X X eleutheraense Bartsch, 1946 Opisthosiphon UF 118996 Paso de los Paredones, X paredonense Torre & Cuba Henderson, 1921 Opisthosiphon simpsoni UF 42775 Abaco Island, Bahamas X Bartsch, 1946 (1) Opisthosiphon simpsoni GTW Andros Island, X X Bartsch, 1946 (2) 14188b Bahamas Parachondria canescens UF 119113 Eleuthera, Bahamas X (Pfeiffer, 1852) (1) Parachondria columnus UF 249219 Middlesex, Jamaica X X (Wood, 1828) Cont.

13 Table 1 Cont. Parachondria crenulocom Jamaica X X X Parachondria fascius UF 218163 High Gate, Jamaica X (Wood, 1828) Parachondria fecundum Jamaica X X X Parachondria fecundum Jamaica X X X (2) Parachondria graminosus UF 218041 Ponce, Puerto Rico X X (Baker, 1941) Parachondria lineolatus UF 128918 Tortola, Virgin Islands X X X (Lamarck, 1822) Parachondria lineolatus UF 191196 Great Camanoe, Virgin X X X (Lamarck, 1822) (2) Islands Parachondria mutica Jamaica X X X Parachondria pyrostoma UF 218203 San Andrés (off Nicaragua) X Pilsbry, 1930 Parachondria rubicundus UF 290100 Biósfera Tawahka- X X X (Morelet, 1849) Asangi, Honduras Parachondria wilkinsoni X Ramsdenia garciana GTW 15041a Los Tibes, Cuba X X X Aguayo, 1932 Ramsdenia zayasi GTW 15044a Sao Corona, Cuba X X X Jaume, 1984 (2) Ramsdenia zayasi UF 119107 Cape San Lucas, Cuba X X Jaume, 1984 (2) Rhytidopoma torreianum UF 160956 Ceiba Mocha, Cuba X X X (Arango y Molina, 1878) Rolleia martensi UF 249224 Plaisance, Haiti X X (Maltzan, 1888) Tudora aurantia (Wood, UF 119133 Curaçao X 1828) Tudora megacheilos UF 119126 Curaçao X (Potiez & Michaud, 1838) (1) Tudora megacheilos GTW 7289e Curaçao X X X (Potiez & Michaud, 1838) (2) Tudora rupis Baker, 1924 GTW 7237d Curaçao X X X Tudorina rangelina UF 119096 El Retiro, Cuba X X (Poey, 1851) Tudorisca alba (2) Jamaica X X X Tudorisca alba (Sowerby, UF 249200 Runaway Caves, X X X 1843) Jamaica Cont. 14 Table 1 Cont. Tudorisca albus Jamaica X X X Tudorisca bronnii UF 249201 Elgin Hall, Jamaica X X (Adams, 1845) Tudorisca Jamaica X X humphreysiana Tudorisca redfieldiana Jamaica X X X Tudorisca rosenbergiana UF 284131 Grand Cayman Island X X X (Preston, 1911) (1) Weinlandipoma Coll. J. Cavalier, Haiti X X X gonavense (Weinland, Grego 1880) (1) Weinlandipoma UF 48134 Plaine Sa Wo, Haiti X smithianum (Pfeiffer, 1866) Wrightudora garridoiana UF 136575 Las Yaguas, Cuba X X (Torre & Bartsch, 1941) Xenopoma aguayoi GTW 9435b Cantera de Mella, Cuba X X X (Torre & Bartsch, 1941) Xenopoma GTW 9436d Cueto, Cuba X X X spinosissimum (Torre & Bartsch, 1941)

Phylogenetic analyses

Results of the phylogenetic analyses were congruent between the subset and complete data set and across the parsimony, maximum likelihood and Bayesian reconstructions based upon visual inspection of topologies. Each of the major clades were consistently reconstructed, but the differences in relationships occurred at the basal nodes, resulting in polytomies in the strict consensus of the parsimony tree and the majority rule posterior tree. Figure 2 is the maximum likelihood reconstruction with bootstrap ≥ 70 and posterior probability ≥ 90 clades indicated. Monophyly of the

Annulariidae was strongly supported in analyses of both data sets (bootstrap ≥ 90 and posterior probability ≥.95), and in all trees reconstructed resulted in Annulariidae being

15 sister to the Pomatiidae. In analyses using the full data set, Pomatiidae was recovered as monophyletic, making Annulariidae and Pomatiidae sister with respect to the Littorinidae outgroup.

In general, the reconstructions did not show monophyletic groupings of the geographic regions; however, there were general trends within the tree. The most basal lineage (Figure 2, Clade A) are representatives from the genera Abbottella and Lagopoma collected from Hispaniola (non Tiburon Peninsula), which is then followed by a lineage with representatives from primarily Central America (Figure 2, Clade CA). This placement of the Central American taxa was not recovered in the Bayesian reconstruction of the subset analysis (Figure 3), rather it was sister to a clade of Cuban taxa; however, it was recovered in the likelihood and parsimony subset trees.

Figure 2 Maximum likelihood reconstruction of the full data set. Support values are bootstrap replicates (≥70) and posterior probability (≥.9). Branch lengths are scaled.

17 Incorporated into a paraphyletic grade of Cuban taxa, is a monophyletic grouping of species from the genus Opisthosiphon from the Bahamas (Figure 2, Clade O). At the tip of the tree, there are two sister groups with one group primarily Jamaican with specimens from ABC islands, Hispaniola and the Lesser Antilles, and the other group is primarily Hispaniolan with specimens from Jamaica, ABC islands and Cuba (Figure 2,

Clades J and H respectively).

These patterns were seen in the subset analysis using RaxML and TNT. The subset in MrBayes was consistent with the exception of the placement of the Central

American fauna (Figure 3) which is sister to several Cuban taxa as opposed to basal to them.

Figure 3 The output tree from the BBM analysis on the subset data. The topology and support values represent the majority rule posterior tree distribution. The circles represent the ancestral range reconstruction, with the relative proportion predicted.

19 Ancestral range reconstruction

The BBM and S-diva analyses were consistent in their reconstruction of the ancestral ranges, showing the common ancestor of all Annulariidae to be primarily from

Cuba (Figures 3 and 4). S-diva showed more variability at each node for the option of ancestral ranges, but still provided similar support for the ranges determined in the BBM analysis. Figure 3 shows the ancestral reconstructions from the BBM analysis, with the pie charts on each node showing the posterior distribution of the range for that ancestor.

Figure 4 shows the ancestral reconstructions based upon the S-diva analysis, which allows for more possible combinations of ancestral ranges.

The relative role of dispersal, vicariance and extinction at each node was assessed in the S-Diva analysis resulting in 11 dispersal events, 11 vicariance events and no extinction events after excluding the node with the outgroup (Node 75, Figure 4).

Dispersal events occurred at nodes 41, 48, 51, 56, 57, 71, 73 and 74 (Figure 4, nodes indicated with a *) while vicariance events occurred at nodes 40, 47, 50, 53, 56, 59, 68,

70, and 72 (Figure 4, nodes indicated with #). The node representing most recent common ancestor to all Annulariidae was reconstructed as leading to dispersal events

(Figure 4, nodes 73 and 74), and the node at which the separation of the major land mass lineages diverged was reconstructed as a vicariance event (Figure 4, node 72), as was the common ancestor to the Jamaican and Hispaniolan clades (Figure 4, node 59).

Figure 4 Ancestral range reconstruction using S-Diva. Dispersal events are indicated as (*) and vicariance events are indicated with (+).

Discussion

Origins

Based upon the ancestral range reconstruction and the species diversity patterns, it appears that the common ancestor to the Annulariidae came from a Cuban or Central

American landmass, centering around the Yucatan Peninisula and Cuba, and radiating to

Hispaniola and Jamaica in a stepping stone pattern. Following the model of Pindell and

Barrett (1990), landmasses were available by the late Eocene, primarily surrounding

Western Cuba and Central America, and as the Caribbean plate emerged, more land was available for colonization allowing for dispersal to these newer landmasses. While the distance between land grew greater and more niches filled, the probability of dispersal between them decreases, explaining the sequential decrease in species richness.

This pattern presented in these analyses is contrasted with what would be expected under the GAARlandia model, which would imply that the Caribbean taxa originated in South America with subsequent dispersal to the Greater Antilles through the

Lesser Antilles. As seen in Figure 2, Central American taxa are basal to other lineages with the exception of Abbottella (Figure 2, clade A), which are separated from other

Annularids by unique morphological features (Watters et al. in prep). The possible, somewhat continuous distribution of ancestral Annulariidae across the Proto-Antilles with subsequent separation events as the Caribbean plate pushed through North and 22 South America explains the present day pattern as well as the short branch lengths associated with the separation of major lineages (Figure 2).

In a study of another major Caribbean land snail group, the Urocoptids, Uit De

Weerd (2008) came to similar conclusions around the origins of the taxa using 28S nuclear ribosomal marker and the available fossil record. The author argues that the separation of the old world and new world taxa lead to origins of the Urocoptids in upper

North America during the late Cretaceous with subsequent movement to the Antilles through the proto-Antilles. This is consistent with these findings as the presumed closest relative to the Annulariidae, are the old world Pomattidae, suggesting that the origination of the Annulariidae occurred prior to the subduction of the Caribbean plate. The lack of fossil Annulariidae prohibits the ability to further support this claim with conclusions being limited to the availability of the taxa sampled.

Biogeographic patterns

In general, geographic areas did not correspond to monophyletic groupings as would be expected under a strictly vicariant process. The basal lineages include taxa from

Hispaniola, Central America and Cuba, suggesting a continuous distribution of taxa across the Proto-Antilles, centralized around Cuba and Central America. Jamaica and

Hispanola represented two major grades that correspond to a divergence event with subsequent dispersals to other areas (Figure 2 Clades J and H). Clade J was primarily

Jamaican with dispersals to the Hispaniola and the Lesser Antilles, while Clade H is primarily Hispaniolan with dispersals to Jamaica, ABC islands and Cuba.

23 The basal lineage comprising Abbottella and relatives are all present on

Hispaniola, and given their morphological differences, could represent a new taxonomic group within the Annulariidae. Their placement relative to other Hispaniolan taxa suggest that their movement to the region as independent from the other organisms. Under current taxonomic treatment based upon radula morphology, it would have been expected to find the taxa within the genera Meganiphe and Annularisca to be placed within this Abbottella group (Thompson 1978, Watters 2006). However, this was not the case, which suggests issues with taxonomic and genetic sampling, or that radula morphology is more plastic than previously thought.

The other basal lineages are sister goups consisting of a clade based in Central

America (Figure 2, clade CA) and a grade of Cuban taxa. This relationship supports a proto-Antillean origin of the Annulariidae since the western most groups comprise the base of the tree. Within the grade of Cuban taxa, is a grouping representing the genus

Opisthosiphon (Figure 2, Clade O) which is primarily found in the Bahamas. The position of Parachondria canescens nassauensis, at the base of the clade supports the origins of this group from Cuba since it is believed to be an anthropogenic introduction to the Bahamas but appears to be from Cuba (Bartsch 1946, Watters 2006, 2014a).

Cuba has been a “hot-spot” for these snails with much of the diversity of this family originating on the island. Containing about 300 of the approximately 700 species, the Cuban taxa is some of the most diverse and encompasses multiple unique lineages.

The centrality of the island, complex geologic history of being three distinct units and evidence of being at least partially emerged since the middle Eocene (Graham 2003) continues to support this island as a center for Annulariid dispersal. As one of the longer 24 lasting landmasses in the region, it supports the rapid diversification on the island and subsequent dispersals to other landmasses.

There appears to be two independent invasions of Jamaica, which contradicts what studies of other taxa have determined (e.g. in Selenops, Crews & Gillespie 2010; in

Eleutherodactylus, Hedges 1996). The first invasion appears to have occurred at a similar time to the invasion of Hispaniola, whereas the second invasion occurred as a dispersal event from taxa on the Tiburon Peninsula. The taxa from the second invasion of Jamaica are from the genus Parachondria, which appears to be separate from other Parachondria in the Caribbean, making the genus polyphyletic (Figure 2, Clade H).

Temporal relationships

The weak internal branch support for each of these analyses is characteristic of deep, rapid divergences followed by subsequent radiations and has been a characteristic of other taxa within this region (e.g. Crews & Gillespie 2010). The short length of these internal branches (Figure 2) make it difficult to recover a well supported tree and would require extensive sampling of different markers to find variation that occurred during that time period; this is generally unreasonable for most phylogenetic studies of this scale.

Because of this, it is illadvised to use a molecular clock with these data. Temporal information can be gleaned about the relative separation of major geographic lineages and potential mechanisms for the movement of organisms. However, parsing historical biogeographical relationships are further challenged by the incomplete history of the availability of landmasses for colonization. Even being able to appropriately date a divergence event, it is difficult correspond the information to the geologic record.

25 Work done by Goodfriend and Mitterer has found that terrestrial snails within this region have had long spans of endemism, remaining in their locality for thousands of years (Goodfriend & Mitterer 1988, 1993). They suggest that many terrestrial snails, primarily in Jamaica, have persisted since the late Pleistocene to the present (Goodfriend

& Mitterer 1988, 1993). The implications of their work on this study provides support for a period of rapid radiation and dispersal, followed by a prolonged period of limited diversification which could have been constrained by their ability to disperse (Goodfriend

& Mitterer 1993) or by the lack of available niches (Goodfriend 1989). Even though their work emphasizes Jamaica and more recent dates of organisms around the Holocene, the long tip branches in this study suggest that many of the main lineages within

Annulariidae have persisted and resulted after the separation and emergence of the major landmasses.

Evidence for dispersal

As described in Watters (2014), the general trends of the Annulariidae within the

Lesser Antilles suggests that the lower lesser Antilles have a different set of taxa and origins than the higher islands, as seen in multiple other taxa (eg. Losos & Thorpe 2014).

In Figure 3, it can be seen that the taxa of the ABC islands appear to have arrived from

Cuba independently of the upper Lesser Antilliean islands. Under both the GAARlandia model or a strict vicariance model, it would be predicted that the Lower and Upper Lesser

Antilliean fauna would be more closely related and have shared origins. This was not recovered in any of the analyses. However, it is possible to invoke the GAARlandia land bridge as an explanation for the arrival of the snails to the ABC islands since for a period

26 of time there would be a change in oceanic currents. The change in currents would provide an opportunity for rafting from Cuba to the south.

These reconstructions show strong evidence for over-water dispersal in part as a mechanism to describe the distributional patterns. There appears to have been two separate colonizations of Jamaica from other parts of the proto-Antilles with subsequent dispersal to the Lesser Antilles and the ABC islands. The initial colonization occurs with the separation from parts of Hispaniola, whereas the second colonization appears to be the result of over-water dispersal. This process also explains the origins of organisms to the Cayman islands, which must have resulted from dispersal via rafting or other mechanisms as seen in the presence of Tudorisca rosenbergiana in the clade of Jamaican fauna (Figure 2, Clade J).

Challenges and future directions

Further studies using increased taxon and genetic sampling could provide more information on the relationships between major clades that is necessary to make stronger assessments of the working hypotheses. We were limited by the availability of specimens and certain areas did not have specimens available, particularly from Western Cuba due to the inability to collect there or obtain specimens from museums or collectors. Further sampling in Cuba could show more diversity or geographic structure than previously thought on the island as well as provide a link between the Yucatan and Cuban taxa. In the subset analysis, the lack of good sequences from Central America emphasized Cuba as the origin of this family but further sampling in the Yucatan could alter this interpretation. The impact of this missing geographic area is unknown but sampling this

27 area could further support the origins of these taxa. Work incorporating morphological data may elucidate some confounded relationships amongst taxa.

Extinction has potentially played a large role in shaping the Caribbean fauna.

With the complex history of each of the island, many of which had turbulent pasts of submergence and emergence, it is not a surprise to find the heterotachy as seen in this phylogeny (Figure 2). Without fitting a model of molecular clock and having short branch lengths resulting in polytomies at the basal nodes, it was not possible to directly assess extinction using a DEC model as implemented in the programs such as

LAGRANGE.

Conclusion

This study is one of the most broadly sampled work on the biogeography of the

Caribbean and provides some clarification into the processes leading to the distributional patterns observed within Caribbean taxa. There is strong evidence of a joint over water dispersal and vicariance model to explain the relationships of the Annulariidae, although there is little evidence for GAARlandia. The concordance of this taxa with others throughout the region gives further support for current explanations and provides a basis for further comparative biogeography.

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