The Pennsylvania State University
The Graduate School
Eberly College of Science
CRYPTIC DIVERSITY, EVOLUTION, AND BIOGEOGRAPHY OF CARIBBEAN CROAKING GECKOS (GENUS: ARISTELLIGER)
A Thesis in
Biology
by
Tiffany Loren Cloud
© 2013 Tiffany Loren Cloud
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Master of Science
May 2013
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The thesis of Tiffany Loren Cloud reviewed and approved* by the following:
S. Blair Hedges Professor of Biology Thesis Adviser
Charles R. Fisher Professor of Biology, Assistant Department Head for Graduate Education
Tracy Langkilde Associate Professor of Biology
*Signatures are on file in the Graduate School.
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Abstract
The sphaerodactylid gecko genus Aristelliger (Caribbean croaking geckos) currently contains eight described species, all found on Caribbean islands. Mitochondrial DNA (mtDNA) sequence data from 106 specimens representing all eight currently recognized species were used to evaluate the relationships of the species within the genus. The molecular phylogeny of the genus is inconsistent with the current species-level taxonomy. Species delimitation methods indicate that there are as many as 24 species, hidden within the eight currently described species. This was especially true of Aristelliger praesignis (Jamaican Croaking Gecko) which has 10 clades of potential species-status, eight of which are in Jamaica, showing deep genetic divergences. Further support that at least six of these clades represent cryptic species comes from the consistency with which most clades can be differentiated from each of the other clades based on morphological characters. In addition, two clades are sympatric and syntopic at Port
Antonio, Portland Parish, Jamaica. Another two clades are nearly sympatric, separated by only about 0.75 km at Port Maria and Cabarita Island, St. Mary Parish, Jamaica. In addition to the high levels of cryptic diversity found within Jamaica, A. georgeensis (St. George Island
Gecko)—the only member of this genus found on the mainland—is nested within a clade of A. praesignis from Port Antonio, and is the most recent of the clades to diverge, about 1.5 million years ago (Ma). Based on molecular divergence time estimation and geology, it is likely that this genus originated on Hispaniola between 37 Ma and 23 Ma. From there, a lineage invaded
Jamaica giving rise to A. praesignis. There have been three dispersals out of Jamaica giving rise to populations or species elsewhere: two out of northern Jamaica, and one very recently from southwestern Jamaica.
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Table of Contents
List of Figures……………………………………………………………………..…..………...v
List of Tables……………………………………………………………………………..….….vi
Introduction …………………………………………………………………………………...... 1
Materials and Methods………………………………………………………………..…...... 11
Results…………………………………………………………………………………….…....17
Discussion………………………………………………………………………………….…..27
References……………………………………………………………………………………..37
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List of Figures
Figure 1—Bayesian inference tree showing relationships within Aristelliger...... ……………...…20
Figure 2— Maximum likelihood tree showing relationships within Aristelliger ……………….....21
Figure 3— Portion of maximum likelihood tree showing relationships within A. praesignis with a map of Jamaica showing the localities of the Jamaican clades……………………....22
Figure 4—Molecular timetree for the genus Aristelliger………………………………………...…..23
Figure 5—Neighbor Joining tree showing groups defined by ABGD………………..……...... ….24
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List of Tables
Table 1— Most useful traits for distinguishing between each pair of clades……………………..25
Table 2—Weighted and Unweighted Classification Scores…………………...……..………….....26 1
Introduction
The incorporation of molecular techniques in taxonomy has revolutionized species discovery (Bickford et al., 2007; Nagy et al., 2012). Two of the past five years have been record-breaking for the number of reptile species described in a single year:
166 species in 2007 and 168 species in 2012 (Uetz, 2013). There were only 3 other years in history that there were over 100 species of reptiles described: 1758, 1854 and
1863 (Uetz, 2013). In addition, 3,076 species of amphibians have been described since
1985, a 75% increase (AmphibiaWeb), and 408 new species of mammals were discovered between 1993 and 2008, a 10% increase (Ceballos & Ehrlich, 2009).
The identification of cryptic species has played a major role in the increased rate of species discovery. Cryptic species are species that have been lumped under one species name due to being, at least superficially, indistinguishable morphologically
(Beheregaray & Caccone, 2007; Pfenninger & Schwenk, 2007; Barata, Carranza &
Harris, 2012). The use of molecular data has been responsible for many of these discoveries. Before 1989, when PCR was first used in evolutionary biology (Kocher et al., 1989), very few studies mentioned cryptic species. However, the number of such studies has risen exponentially since then and by 2005 over 20% of studies mentioned cryptic species (Bickford et al. 2007). Within mammals, 60% of the new species are considered cryptic species (Ceballos & Ehrlich, 2009). Even charismatic, widely studied organisms have been shown to harbor cryptic species, such as giraffes (Brown et al.,
2007), elephants (Roca et al., 2001), and hammerhead sharks (Pinhal et al., 2012).
Furthermore, cryptic species are not limited to a certain region or environment and have been found in developed countries that have been well-studied such as Australia,
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Europe, and North America (Kiefer & Veith, 2001; Rissler & Apodaca, 2007; Oliver et al., 2009; Manthey, Klicka, & Spellman, 2011; Smith et al., 2011; Yang et al., 2012).
There are certain natural history traits that seem to be associated with having higher levels of cryptic diversity. Organisms that are less dependent on visual cues in selecting mates, using instead nonvisual mating cues such as acoustic calls or pheromones tend to have a greater number of cryptic species (Bickford et al. 2007;
Hoskin & Higgie, 2010; Clare et al., 2011). In addition, certain environmental factors can impose strong selection on maintaining the ancestral morphology. For example, symbionts specialized to specific host tend to harbor cryptic species due to strong selection to maintain the traits that enable them to infiltrate the host’s body or society
(Schönrogge et al., 2002; Locke, McLaughlin, & Marcogliese, 2010). Extreme environments, such as; subterranean, arctic tundra or deep sea environments also tend to harbor greater numbers of cryptic species (Lefébure et al., 2006; Bickford et al.,
2007; García-Machado, 2011).
Genus: Aristelliger
The genus Aristelliger, commonly known as the Croaking Gecko or the
Caribbean Gecko, is currently comprised of eight recognized species (Diaz & Hedges,
2009). The members of this genus are found exclusively in the Caribbean and along the east coast of Central America (Bauer & Russell, 1993a). The genus is characterized by the following morphological characteristics: skin that is easily torn, mottled with browns and tans, small granular scales, vertical pupils, friction pads on at least two digits, bones in the hemipenes, oil droplets in rods, undivided lamellae, croaking call, and all
3 digits having claws (Cope,1862; Underwood, 1954; Bauer & Russell, 1993a; Diaz &
Hedges, 2009).
Members of this genus are primarily arboreal. They are often found in trees; living, dead, or rotting and often under the bark. They are commonly associated with
Coconut Palms (Cocos) and Fig trees (Ficus) but this varies by species. In addition, they are found in palm trash and under rocks (Noble & Klingel, 1932; Thomas, 1966;
Schwartz & Crombie, 1975; Henderson & Powell, 2009). They also take advantage of man-made structures, such as on thatched roofs, in rafters, in crevices of walls or on fences (Schwartz & Henderson, 1991; Lee, 1996; Henderson & Powell, 2009). At night they are quite vocal (Dunn & Saxe, 1950; Hecht, 1952; Schwartz & Henderson, 1991).
Their diet is primarily composed of arthropods (Henderson & Powell,1999), though
Aristelliger cochranae females are known to eat gecko eggs and hatchlings (Gifford et al., 2000), A. georgeensis—anoline lizards (Dunn & Saxe, 1950; Lee, 1996) and A. lar will eat berries and flowers (Burns et al., 1992; Henderson & Powell, 2009), in addition to arthropods. Females lay a single egg per clutch (Kluge, 1967; Daza & Bauer, 2012) up to twice a year (Hecht, 1952). The eggs are typically sticky and are laid on trees; sometimes in the open or in hollows and under the bark; in crevices between rocks; or on the backs of fronds (Henderson & Powell, 2009). The eggs can be laid singly or in a communal nest and are incubated for about 3 months (Barbour, 1910; Noble and
Klingel, 1932; Hecht, 1952).
There are two subgenera within Aristelliger distinguishable by size and number of friction pads. The smaller subgenus is Aristelligella (Noble and Klingel, 1932), which has a maximum snout-vent length of 63 mm and friction pads on three fingers and two toes.
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The subgenus Aristelliger (Cope, 1862) is much larger, with a maximum SVL of 135 mm. They have friction pads on only one finger and one toe (Hecht, 1951; Bauer &
Russell 1993a; Diaz & Hedges, 2009).
Subgenus: Aristelligella
Between 1931 and 1933 three of the four species belonging to the subgenus
Aristelligella were described. The first to be described was Cochran’s croaking gecko,
Aristelliger cochranae, which was described in 1931 by Grant. This species is found on
Navassa Island off the coast of Haiti. Grant named it in honor of Doris Cochran who was the curator of herpetology at the US National Museum at that time (Grant, 1931;
Thomas, 1966; Lynxwiller & Parmerlee Jr, 1993).
In 1932, Noble and Klingel described a new species of gecko from the Inagua
Islands in the Bahamas. They named this gecko, Aristelligella barbouri (Striped
Caribbean gecko), in honor of Thomas Barbour, who was director of the Harvard
Museum of Comparative Biology at that time. They erected Aristelligella as a genus unique from Aristelliger based on differences in the plates adjacent to the claw on some digits. They assigned two species to this genus, A. cochranae and A.barbouri, with A. barbouri as the type species (Noble and Klingel, 1932; Bauer & Russell, 1993b). Twenty years later Hecht suppressed A. barbouri to a subspecies of A. cochranae and acknowledged Aristelligella as a subgenus of Aristelliger with its only species being A. cochranae with its 3 subspecies (Hecht 1951, 1952). Aristelliger barbouri was recognized as a full species again by Schwartz and Henderson (1991), and Bauer and
Russell (1993b).
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The following year, 1933, Cochran described a new species from Haiti and southwestern Dominican Republic. She named it Aristelliger expectatus due to a prediction made by Noble that there should be a species that was closely related to A. cochranae in Haiti (Cochran, 1933). Its common name is the Hispaniolan Desert Gecko.
In 1939, Mertens reduced its status to being a subspecies of A. cochranae with no explanation (Lynxwiller & Parmerlee Jr, 1993). This status was maintained until 1996 when it was recognized as a full species based on morphology and the difficulty of gene flow between Navassa Island and the mainland of Hispaniola (Powell, 1996).
The fourth species in this subgenus, Aristelliger reyesi (Reyes Caribbean
Gecko), was recently described by Diaz and Hedges in 2009. This was a surprising discovery because the only locality it is known from is Peninsula de Hicacos in western
Cuba which is close to a popular center for tourism. The authors named the species in honor of Ernesto Reyes who took the photo in 2007 that lead to the discovery of this species (Diaz and Hedges, 2009).
Subgenus: Aristelliger
The type species for the genus and subgenus is Aristelliger lar (Hispaniolan
Giant Gecko or Spotted Caribbean Gecko). It is the largest species of Aristelliger reaching a maximal size of 135 mm SVL in males and 111 mm SVL in females. The species was described in 1862 by Cope, and is sparsely distributed in Hispaniola. It was probably named after a Roman household deity likely in reference to the first specimen being collected in a house (Bauer & Russell, 1993c).
Aristelliger praesignis (Croaking gecko or Woodslave) was described by
Hallowell in 1856 as Hemidactylus praesignis, and was placed in the genus Aristelliger
6 by Cope in 1862. There are two subspecies, the nominate subspecies, A. praesignis praesignis, is found in Jamaica and the Cayman Islands. The second subspecies, A. praesignis nelsoni, is found on the Swan Islands off the coast of Honduras (Schwartz &
Henderson, 1991; Bauer & Russell, 1993d). It was first described in 1914 by Barbour, as A. nelsoni; however, in 1951, Hecht reduced it to subspecies status.
Only one species in the genus is found outside of the West Indies, Aristelliger georgeensis (Bocourt, 1873: St. George Island gecko). It is found on the mainland in
Belize and Mexico as well as on several islands off the Atlantic coast of Central America
(Bauer & Russell, 1993e). They make a loud screeching call which has earned them another common name in Belize, the weatherman, because it apparently makes this call before a storm (Dunn & Saxe, 1950; Schwartz & Henderson, 1991). This species was described in 1873 by Bocourt, as Idiodactylus georgeensis named for its type locality,
St. George’s Island. Aristelliger georgeensis has had a complicated taxonomic history. It was moved into the genus Aristelliger by Cope in 1885 when he described a population from Cozumel, Mexico as a different species, A. irregularis. The same year it was synonymized with A. praesignis (Boulenger, 1885). In 1941, Schmidt synonymized the species described by Bocourt (1873) and Cope (1885) using the name A. georgeensis.
A fourth member of the subgenus, Aristelliger hechti (Hecht’s gecko), was discovered in 1951 by Hecht. However, it wasn’t formally described until 1975
(Schwartz & Crombie). The species was named in honor of Max K. Hecht. This species is restricted to the Caicos Islands (Schwartz & Henderson, 1991; Bauer & Russell,
1993f) but it has also been suggested that it is in Northern Haiti, but misidentified as A. lar (Burns et. al, 1992; Powell & Parmerlee, 1992; Lever, 2003).
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Establishing relationships between genera and families of geckos has been highly controversial and many phylogenies have been proposed (Underwood, 1954;
Kluge, 1967, 1982, 1987; Hans et al. 2004; Gamble et al., 2008; Daza & Bauer, 2012).
Many phylogenies have been based on very few morphological traits such as pupil shape, eyelids, cloacal bones digital morphology, and skeletal characteristics
(Underwood, 1954; Kluge, 1967, 1982, 1987; Russell, 1972; Daza & Bauer, 2012). In most of these phylogenetic hypotheses the genus Aristelliger has stood out as being particularly difficult to place (Russell & Bauer, 1993a; Hedges, 1996). This has been attributed to the retention of primitive characters (Underwood, 1954). Recently, efforts have been made to resolve the higher level taxonomy of geckos using molecular sequence data (Carranza et al, 2002; Hans et al. 2004; Carranza & Arnold, 2006;
Gamble et al., 2008; Heinicke et al., 2011). This has resulted in the discovery of new groups of genera that had previously never been associated. One such discovery was the grouping of Aristelliger, Euleptes, Quedenfeldtia, Pristurus, Saurodactylus and
Teratoscincus. This group forms a clade that is sister to the sphaerodactyl clade and therefore the group has been incorporated into the family Sphaerodactylidae (Gamble et al., 2008). No previous phylogenetic hypothesis has included Aristelliger in this family though similarities have frequently been noted (Barbour, 1914; Underwood, 1954; Hass,
1991; Hedges, 1996). Aristelliger’s position within this clade is still not well resolved.
Molecular data supports Quendenfeldtia as the sister genus to Aristelliger (Gamble et al., 2008); while a recent morphological phylogeny strongly supports a sister relationship between Aristelliger and Teratoscincus (Daza & Bauer 2012).
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While a few studies have attempted to address Aristelliger’s position within geckos (Underwood, 1954; Kluge, 1987; Gamble et al., 2008; Daza & Bauer 2012), very little work has been done to address how the species in this group are related to each other (Hedges, 1996). In 1952, Hecht recognized two subgroups within the genus,
Aristelliger and Aristelligella. Schwartz and Crombie (1975) speculated that A. hechti was probably more closely related to A. lar based on trends of zoogeography of the
West Indies. In addition, a close relationship of A. cochranae, A. expectatus and A. barbouri was expected. There is also some debate about the identification of one of populations of Aristelliger in Northern Hispaniola. Traditionally they have been identified as A. lar. However it has been suggested that the populations in the north might be A. hechti instead (Burns et. al, 1992; Powell and Parmerlee, 1992 and Lever, 2003).
In addition, it has been suggested that multiple species may exist within previously described species, especially Aristelliger praesignis, A. georgeensis and A. lar (Schwartz & Crombie, 1975). Crombie (1999) states the reason and frustration behind this suggestion well: “The genus is remarkably conservative and characters to distinguish between unquestionably different species are few, so analysis of intraspecific variation is meaningless using traditional meristic and morphological traits.”
Within Jamaica, the range of lamellae counts of adults and juveniles, average
SVL, and minimum breeding size have been shown to vary between localities (Hecht
1952). However, he attributed these differences to differential selection and small sample sizes and criticized the interpretation that these differences could suggest the presence of multiple species or subspecies of Aristelliger praesignis. It has also been
9 noted that A. praesignis praesignis from Jamaica and the Cayman Islands differ in appearance (Grant, 1940; Crombie, 1999).
The use of molecular data has often been useful in clarifying systematic problems in taxa that have been difficult to resolve based on morphology due to traits that are highly variable within and between species, or groups that maintain their ancestral morphology (Carranza & Arnold 2006). No previous comprehensive phylogenetic hypotheses have been presented for the relationships of the species within this genus (Hedges, 1996; Crother, 1999). A few tentative suggestions have been proposed for relationships of a few species within this genus (Hecht, 1951; Schwartz &
Crombie, 1975). In this study we utilize mitochondrial DNA (mtDNA) sequence data from 106 specimens representing all eight currently recognized species to evaluate the relationships of the species within the genus. This provides a framework to 1) evaluate the monophyly of the described species 2) determine species boundaries and provide taxonomic resolution for species whose status has been unstable 3) provide insight into whether populations of Aristelliger hechti are present in Hispaniola and being misidentified as A. lar (Burns et. al, 1992; Powell & Parmerlee, 1992; Lever, 2003) 4) elucidate the historical patterns of evolution and dispersal within this genus.
Another major objective of this study was to determine if cryptic species were present in this group as previously suspected (Schwartz & Crombie 1975). We focus most of this effort on Aristelliger praesignis praesignis from Jamaica and the Cayman
Islands. Potential cryptic species were identified using Automated Barcode Gap
Discovery (ABGD: Puillandre et al., 2011). This method avoids problems associated with basing hypothetical species discovery on purely tree based methods by making
10 predictions of species richness independent of taxonomic names, while also generating a threshold value that is based on the dataset rather than relying on a generic threshold to identify species (Collins & Cruickshank, 2012). We also performed preliminary morphological analyses on the Jamaican species, lending additional support to the validity of the hypothetical species generated.
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Materials and Methods
Specimen collection and preservation:
West Indian specimens used in this study were collected by hand by Dr. B.
Hedges using protocols approved by the Penn State Institutional Animal Care and Use
Committee. Tissues were deposited in a -80 degree ultracold freezer and voucher specimens were prepared using ethanol fixation for future morphological comparative analyses. Additional tissues and specimens were obtained from other researchers or borrowed from museums. A total of 106 individuals representing all eight species were included in the molecular analysis. While specimens were collected from throughout the
West Indies and also from Honduras, sampling efforts were particularly focused on
Jamaica to evaluate the potential for cryptic species within Aristelliger praesignis.
Molecular Analyses:
Three mitochondrial genes: Cytochrome B (Cytb), 12s ribosomal RNA (rRNA) and 16s rRNA were sequenced for this study. The 12s and 16s rRNA genes were chosen because of their slower rates of evolution compared to other mitochondrial genes and Cytb was chosen to provide finer resolution of the clades and for its usefulness in DNA barcode analyses. The final concatenated dataset contains 3,168 aligned nucleotide sites. Sphaerodactylus was used as the outgroup in our analyses because of its close relationship to Aristelliger (Gamble et al., 2008), as well as the scarcity of molecular data for the genes we analyzed for Aristelliger’s closer relatives
(Gamble et al., 2011).
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DNA extraction, Amplification and Sequencing:
Whole genomic DNA for all individuals was extracted from fresh or frozen tissue using the Puregene tissue kit following the manufacturer’s protocol (Qiagen). An 812 bp fragment of the Cytb gene was amplified using the primer pair SphaeroCytB_612L and
SphaeroCytB_612H. The 12s rRNA and 16S rRNA sequences were generated using overlapping sequences from 5 primer pairs: 12L2/12H3, 12L17/12H11, 12L11/16H27,
16L16/16H7and 16L5/16H25 resulting in a 2,477 bp fragment. The 12s and 16s primers were obtained from previous studies (Hedges, 1994; Feller & Hedges, 1998;
Adalsteinsson et al., 2009). The Cytb primers were designed in lab for greater specificity to sphaerodactyl geckos (This study). The primer sequence for SphaeroCytB_612L is:
AAC YRC YGT TGT WAT TCA ACT A. The primer sequence for SphaeroCytB_612H:
GCN GGK RTR AAR TTT TCT GGG TC.
PCR reactions (25 ul) were performed using the following thermocycler profile: an initial denaturation period of 94°/2.5 min, followed by 40 or 45 cycles of denaturation at 94°/30s, annealing at 50° or 55°/45s; an extension at 72°/45s; followed by a final extension at 72°/7 min. PCR amplification success was verified using UV visualization on a 2% agarose gel stained with Ethidium bromide. Excess primer, dNTP’s and other impurities were removed using gel purification using the Ultrafree-DA gel filters
(Millipore). The purified samples were then sent to the Penn State Genomics Core
Facility - University Park, PA for automated DNA sequencing. Both the forward and reverse strands were sequenced using the same primers that were used for DNA amplification, and run on an ABI 3730XL sequencer.
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To check for call errors the forward and reverse sequences for each sample were compared to each other with reference to their chromatograms. Ambiguities were edited, a consensus sequence was formed, and primer sequences were removed.
Sequences for all primer pairs were aligned and analyzed separately in MEGA 5.05
(Tamura et al., 2011) to check for errors before the data were assembled. Sequences were aligned using MUSCLE (Edgar, 2004) initially, and refined by eye with reference to the amino acid translation. Positions where homology could not be determined were excluded from subsequent phylogenetic analyses.
Phylogenetic Analyses:
Maximum Likelihood analysis was performed on the concatenated dataset containing all three genes using RAxML 7.2.8 (Stamatakis,Hoover, & Rougemont,
2008). The data were partitioned by the combined 12s and 16s rRNA genes, and by gene and codon positions for Cytb. The GTRGamma model was used in the search for the best tree. Remaining parameters were calculated during the run. Gaps were treated as missing data.
In addition, a partitioned Bayesian Inference analysis was performed in MrBayes v3.2.1 (Huelsenbeck et al., 2001; Ronquist & Huelsenbeck, 2003 Ronquist et. al., 2012) using the GTR+I+G substitution model. The 12s rRNA and 16s rRNA genes made up a single partition. Cytb made up the other partition and each site was analyzed independently. The analysis was run for 5,000,000 generations with 4 chains, 3 heated chains and 1 cold chain which were sampled every 100 generations. The first 20% of samples were discarded as burnin; plots of log likelihood vs. generation were generated
14 to ensure that a sufficient number of samples had been discarded. Both analyses were conducted using CIPRES (Miller, Pfeffer, & Schwartz, 2010).
Divergence time estimate:
Divergence times and molecular timetree estimations were conducted using
Bayesian methods. Many popular programs used to estimate molecular timetrees use one of two types of models; a random (independent) rate model or an autocorrelated rate model. It has been shown that typically the results of these two models differ and there are currently no tests available that are powerful enough to accurately predict the appropriate model to use (Battistuzzi et al., 2010). For this reason we used
MCMCTREE (Yang, 2007), which can perform both models. The results from each model were combined to provide more accurate time estimation.
Calibrations used in this analysis were based on geologic history of the West
Indies, as well as divergence times obtained from previous studies. Fossils were not used because all fossil remains belonging to this genus are recent, no older than the
Pleistocene (Hecht, 1951). The maximum divergence time for the genus was set to 37.2
Ma. This date corresponds to the Greater and Lesser Antilles becoming permanently subaerial, based on geologic evidence (Iturralde-Vinent & MacPhee, 1999). The second calibration provides a maximum divergence time for the Jamaican clades of 10 Ma.
There is no evidence that Jamaica had permanently subaerial land before this date
(Donovan 2002; Mitchell 2004; Donovan & Paul, 2011). This calibration was applied to the node containing all Aristelliger praesignis as well as A. georgeensis. The third calibration was a fixed calibration based on the divergence time for the split between A. lar and A. praesignis. This provided a minimum divergence time for this group of 4.9 Ma
15 and a maximum divergence time of 14.3 Ma (Gamble et al. 2011). This was applied to the node containing all members of the subgenus Aristelliger.
Species Delimitation:
The Automated Barcode Gap Detector Program (ABGD), accessible at http://wwwabi.snv.jussieu.fr/public/abgd/, was used to identify potential species. It does this by identifying the barcode gap which is the difference between splitting events within species and those between species. The program defines this as the first significant gap after the confidence interval for intraspecific divergence. It then uses this limit to further partition the data until no more gaps are detected (Puillandre et al, 2011).
A pair wise distance matrix was generated in MEGA using the maximum composite likelihood model for the in-group Cytb dataset. This matrix was then uploaded into the
ABGD program and the program was run using the preset parameters.
Morphological Analysis:
Data Collection:
All measurements were taken with calipers that are accurate to 0.05 mm. We took 13 measurements of body proportions, as well as lamellae counts. The height of the rostral scale (RH) measured from the base to the highest point of the scale, height of the suture (SH) between the rostral scale and the first labial measured to the highest point with respect to the rostral scale, and toe width (TW) was measured across the widest point of the 4th toe. The measurements for these three traits were taken both sides and the two measurements were added together. We measured the length (EL) and width (EW) of the ear, EL was defined as the longest diameter of the opening of the ear opening and EW was the widest distance across the opening of the ear across the
16 other axis. We measured snout-vent length (SVL) from the tip of the snout to the opening of the vent. The distance from the eye to the naris (EyN) measuring from the anterior margin of the eye to the naris, ear and naris (EN) distance was measured from the anterior opening of the ear to the naris. The distance between the posterior portion of the eye and the anterior portion of the ear (EE) was measured, the distance between the nares (IN) was also measured. The width of the head (HW) was measured across its widest point, mental width (MW) was measured across the posterior suture of the mental scale, and the diameter of the eye (ED) was measured across the width of the eyeball. Lamellae (LA#) were counted for all digits, and are reported as a sum of these counts.
Data Analysis:
We performed reverse stepwise Discriminant Function Analysis (DFA) to identify traits that were important in discriminating between the clades defined by the molecular data. This analysis was performed using statistiXL in Microsoft Excel. All counts and measurements were included in the analysis and each was scaled by the SVL. A total of
80 specimens were included in this analysis all of which were from localities included in the molecular analyses. Specimens were borrowed from the Museum of Natural History of the University of Kansas (KU) and the United States National Museum of Natural
History (UNMH). In addition, seven specimen of Aristelliger georgeensis from San
Andres Island, Columbia and 10 from Quintana Roo, Mexico were also analyzed. These specimen were borrowed from the Natural History Museum of Los Angeles County
(LACM), University of Colorado Natural History Museum (UCM), and University of New
Mexico Museum of Southwestern Biology (MSB).
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Results:
Phylogenetic Analyses:
The results of the Bayesian Inference and Maximum Likelihood analyses were congruent and the relationships within the genus were well supported (Fig.1, Fig. 2).
The analyses revealed two main groups corresponding to the subgenera, Aristelliger and Aristelligella. Within the subgenus Aristelliger, there was another deep split separating A. lar and A. hechti from A. praesignis. The clade containing A. praesignis was then split into two major clades. One, containing clades A and B which are found on the southern coast of Jamaica, and another containing the north coast clades C-F as well as a clade from Cayman Brac, a clade from Little Cayman and another containing
A. georgeensis (Fig.3).
The results from the phylogenetic analyses for the other subgenus, Aristelligella, reveal complex relationships within this group. There is moderate support for a close relationship between Aristelliger reyesi and A. cochranae. Aristelliger expectatus is paraphyletic and forms a complex of species. This complex includes the three other recognized species in this subgenus. This group (Aristelligella) is currently being revised, taxonomically, and will not be discussed further here.
Divergence time estimates:
The results from the two molecular timetree analyses differed only slightly, by an average of 0.45 Ma, depending on the model used (auto-correlated or independent).
The single timetree presented here (Fig. 4) uses the means of the two analyses. The two subgenera diverged around 22.5 Ma. The earliest spit within the subgenus
Aristelliger, occurred 13.1 Ma between Aristelliger lar + A. hechti and A. praesignis.
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Within Jamaica, a split occurred between the Northern and Southern coast clades about
7.9 Ma. Most of the clades within A. praesignis arose between 3.5 Ma and 4.5 Ma, with the exception of Clade C, which became isolated around 6.7 Ma and E, F+ A georgeensis which split more recently. Aristelliger lar and A. hechti split around 6.2 Ma.
Within the subgenus Aristelligella, A. reyesi and diverged from A. cochranae 6.3 Ma, and they split from the clade containing A. barbouri 9.2 Ma.
Species Delimitation:
The Automatic Barcode Gap Discovery program (ABGD) revealed the presence of 23–24 candidate species (Fig. 5). The barcode gap spans from 2–5% for CytB. Over the first 6 initial partitions, the model that was chosen for the analysis did not affect the number of groups reported. The prior maximum divergence of intraspecific diversity (P), of 0.0129 was chosen as a conservative estimate of species diversity. Puillandre et al.
(2012) found that at P=0.01 the groups identified by the program were very close to those revealed in other studies. At this value the groups defined by the initial partition and the recursive partition differed only in whether Aristelliger georgeensis was included in the group containing Aristelliger praesignis from Portland and Isla Cabarita, Jamaica or whether it constitutes its own unique group separate from its Jamaican relatives.
Morphological Analysis:
Results from the DFA revealed significant morphological differences (p ≤ 0.050) between 20 out of 28 pairs of species that were compared prior to performing a backward stepwise analysis. Following this analysis, only the comparisons between clades A and F; C and D; and C and E revealed no significant differences. Because
DFA requires a complete matrix and many specimens were missing data for the
19 lamellae counts, the missing data (A= 8% B=57% D=31% E=50% F=0%) were imputed based on the mean lamellae counts for each of the genetically defined clades.
Comparisons were performed using the dataset both with and without lamellae counts and results with lamellae were only reported when they increased the accuracy of the comparison. Lamellae counts were not taken for A. georgeensis and the trait was excluded when making comparisons between them and the Jamaican clades. Overall the most useful traits used to distinguish between the clades were: eye diameter (ED) and inter-nares distance (IN), while the least useful traits were ear length (EL) and total lamellae counts (LA#). However, these varied with comparisons. 3 pairs of clades C-E,
C-MX, and E-MX needed only 2 of the 13 traits to classify them with > 90% accuracy, while others required up to 8 of the 13 traits to distinguish between them (Table 1).
Seventeen of the pairwise comparisons correctly classified the clades with ≥95% accuracy while five more were classified correctly with between 85-95% accuracy. Only four pairs of clades were classified correctly less than 80% of the time (A-B: 75%, A-C:
77% A-F: 76% & C-D: 61%) of the time based on the unweighted averages from the jackknife analysis (Table 2).
267929_Negril 100 267931_Negril 267930_Negril 267933_Negril 100 267908_Negril 267910_Negril Figure 1. Bayesian Inference (MrBayes) tree 98 267934_Negril 267941_Negril of the relationships within Caribbean Croak- 267916_Negril 267932_Negril ing geckos based on mtDNA (Cytb, 12s &16s 59 267919_Negril rRNA). Bayesian support values are shown. 267922_Negril 266577_Grand_Cayman 267918_Treasure_Beach 267925_Negril 83 267909_Black_River 100 267917_Black_River 100 100 267938_Black_River 267940_Black_River 267913_Black_River 267947_Black_River 267915_Black_River 267939_Black_River 100 103463_Jackson_Bay 103464_Jackson_Bay 103461_Jackson_Bay 100 103465_Portland_Cave 100 103447_Jackson_Bay 103462_Jackson_Bay 100 100 103589_Catadupa 103466_Portland_Cave 103590_Catadupa 172207_Braeton 100 171613_Braeton 171614_Braeton 101474_Naggo_Head 101481_Naggo_Head 100 171611_Hellshire_Hills 100 171612_Braeton 97 101457_Kingston 267942_Port_Antonio 99 101475_Kingston 91 267948_Kingston 171615_Yallahs 171616_Yallahs 267944_Morant_Point_Lighthouse 100 267945_Morant_Point_Lighthouse 101575_Duncans 172381_Duncans 100 100 96 172379_Duncans 172380_Duncans 171610_Drax_Hall 100 269419_Port_Maria 100 161284_Port_Maria 269418_Port_Maria 100 100 267943_Port_Antonio 79 100 100 267946_Port_Antonio 172378_Isla_Cabarita 100 269378_georgeensis 100 269379_georgeensis 100 269380_georgeensis 62 266498_Little_Cayman 266501_Little_Cayman 100 266499_Little_Cayman 266500_Little_Cayman 100 266580_Cayman_Brac 266583_Cayman_Brac 100 266581_Cayman_Brac 266539_Cayman_Brac 266582_Cayman_Brac 100 160289_lar 160290_lar 102683_lar 98 100 102684_lar 100 267803_lar 266369_hechti 100 98 266584_hechti 90 266588_hechti 100 266589_hechti 266586_hechti 266587_hechti 267580_expectatus 267483_expectatus 95 267597_expectatus 267610_expectatus 100 192646_expectatus 194857_expectatus 100 102644_expectatus 267601_expectatus 100 193003_expectatus 100 100 266238_expectatus 100 194975_cochranae 194974_cochranae 82 83 194976_cochranae 96 268054_reyesi 64 100 268055_reyesi 100 274062_expectatus 104112_expectatus 100 274053_expectatus 0.05 100 192982_barbouri 267614_expectatus 100 100 267632_expectatus 100 267612_expectatus 100 267624_expectatus 100 267631_expectatus 269342_expectatus 267929-Negril 87 267922-Negril 267919-Negril Figure 2. Maximum likelihood (RaxML) 267916-Negril 92 267932-Negril tree of relationships within Caribbean 267908-Negril 74 267930-Negril Croaking geckos based on mtDNA 267910-Negril 267934-Negril (Cytb, 12s &16s rRNA). Maximum 267933-Negril likelihood bootstrap values are shown 61 267931-Negril 267941-Negril for important nodes. 266577-Grand Cayman 267918-Treasure Beach 59267925-Negril 267909-Black River 98 267940-Black River 100 267938-Black River 267939-Black River 267913-Black River 267917-Black River 267947-Black River 267915-Black River 97 103464-Jackson Bay 103463-Jackson Bay 103461-Jackson Bay 100 103465-Portland Cave 89 103447-Jackson Bay 103462-Jackson Bay 100 103589-Catadupa 103466-Portland Cave 103590-Catadupa 171614-Braeton 101474-Naggo Head 101481-Naggo Head 97 171613-Braeton 171612-Braeton 171611-Hellshire Hills 100 172207-Braeton 99 267942-Port Antonio 267948-Kingston 101475-Kingston 99 81 101457-Kingston 171615-Yallahs 171616-Yallahs 267945-Morant Point 99 267944-Morant Point 101575-Duncans 172381-Duncans 100 96 172379-Duncans 78 172380-Duncans 171610-Drax Hall 100 161284-Port Maria 269419-Port Maria 100 269418-Port Maria 100 267943-Port Antonio 98 60 100 100 267946-Port Antonio 172378-Isla Cabarita 83 269378-georgeensis 100 269380-georgeensis 94 269379-georgeensis 70 266498-Little Cayman 266501-Little Cayman 100 266500-Little Cayman 266499-Little Cayman 98 266583-Cayman Brac 266580-Cayman Brac 100 266582-Cayman Brac 266539-Cayman Brac 266581-Cayman Brac 102684-lar 85 102683-lar 160290-lar 100 99 160289-lar 267803-lar 266369-hechti 100 89 266584-hechti 81 266588-hechti 100 266589-hechti 266586-hechti 266587-hechti 267580-expectatus 267483-expectatus 64 267597-expectatus 192646-expectatus 100 267610-expectatus 194857-expectatus 100 102644-expectatus 100 267601-expectatus 99 100 193003-expectatus 266238-expectatus 194974-cochranae 100 194976-cochranae 67 194975-cochranae 92 100 268055-reyesi 56 268054-reyesi 100 274062-expectatus 104112-expectatus 99 100 274053-expectatus 192982-barbouri 0.05 267614-expectatus 100 267632-expectatus 100 100 267612-expectatus 267624-expectatus 100 91 267631-expectatus 269342-expectatus Figure 3. Portion of the ML tree showingthe Jamaican Croaking gecko (Aristelliger praesignis). Each Jamaican clade is color coded as shown on the tree, and the colored dots on the map correspond to the color labels on the tree and represent the localities that each respective clade is found.
267929-Negril 0.765 267941-Negril 267922-Negril 0.332 267908-Negril Figure 4. Timetree of Caribbean croaking geckos 1.0390.2275 (Genus: Aristelliger) using the data and topology of 0.149 267931-Negril 0.0885 267916-Negril the concatenated mtDNA dataset (Cytb, 12s &16s) 1.343 266577-Grand_Cayman data set from the ML analysis (Fig.2). Node labels 267918-Treasure_Beach 267925-Negril represent the divergence times in millions of years 1.0955 0.301 267909-Black_River 267913-Black_River and are averages of the results of two Bayesian 1.662 0.1655 0.0815 267947-Black_River analyses (independent and autocorrelated rates). 0.4615 103463-Jackson_Bay Bayesian credibility intervals (grey bars) are compos- 103464-Jackson_Bay 103461-Jackson_Bay ites of the intervals derived from the two analyses. 0.904 0.2465 103465-Portland_Cave 0.6325 103447-Jackson_Bay 0.1275 103462-Jackson_Bay 0.469 103589-Catadupa 4.479 0.329 103590-Catadupa 0.1495 103466-Portland_Cave 0.379 172207-Braeton 171614-Braeton 0.208 101474-Naggo_Head 0.9275 0.2045 267948-Kingston 7.9295 0.1325 101457-Kingston 0.2975 267942-Port_Antonio 101475-Kingston 3.0515 0.5465 171615-Yallahs 171616-Yallahs 267945-Morant_Point 0.103 267944-Morant_Point 101575-Duncans 0.363 172381-Duncans 6.727 0.1535 172380-Duncans 0.0865 172379-Duncans 171610-Drax_Hall 4.7945 0.1525 161284-Port_Maria 269418-Port_Maria 2.191 0.0985 269419-Port_Maria 13.0825 0.399 267943-Port_Antonio 4.354 172378-Isla_Cabarita 1.4815 269378-georgeensis 0.288 269380-georgeensis 269379-georgeensis 4.1385 0.094 0.0745 266499-Little_Cayman 266500-Little_Cayman 0.1715 266501-Little_Cayman 0.094 266498-Little_Cayman 3.481 0.269 266539-Cayman_Brac 266583-Cayman_Brac 0.0965 266580-Cayman_Brac 0.3915 160290-lar 102684-lar 0.132 102683-lar 1.113 6.2375 160289-lar 22.531 2.272 267803-lar 266369-hechti 266586-hechti 1.315 0.299 266588-hechti 0.1125 266584-hechti 6.673 266238-expectatus 0.6495 193003-expectatus 102644-expectatus 0.0755 267601-expectatus 14.053 104112-expectatus 1.5135 274053-expectatus 7.7185 0.229 194975-cochranae 194974-cochranae 0.078 194976-cochranae 6.298 268054-reyesi 9.1655 2.644 274062-expectatus 192982-barbouri 269342-expectatus 15.794 5.268 0.2275 267614-expectatus 4.424 267632-expectatus 2.566 0.1755 267612-expectatus 267631-expectatus 0.084 267624-expectatus 194857-expectatus 1.02850.141 267597-expectatus 267483-expectatus 0.373 192646-expectatus 0.1905 267610-expectatus
25.0 20.0 15.0 10.0 5.0 0.0 Figure 5. Neighbor-joining tree based on the Cytb dataset showing the groups defined by the Automatic Barcode Gap Discovery program (ABGD). It identified 24 groups, 16 of these represent potential new species.
RH SH EL EW MW TW EyN IN HW ED EN EE LA# A-B X X X X X X X A-B (LA#) X X X X X X X X A-C X X X X A-D X X X X X A-E X X X X X A-E (LA#) X X X X X X A-F X X X X X X X A-F (LA#) X X X X X X A-CO X X X X A-MX X X X X X X X X B-C X X X X X X X B-D X X X X B-E X X X B-F X X X X X B-CO X X X X X X B-MX X X X X X X C-D X X X X C-D (LA#) X X X X X X X X X C-E X X C-E (LA#) X X C-F X X X X C-CO X X X X C-MX X X D-E X X X X X D-F X X X X X X X X D-F (LA#) X X X X X X X D-CO X X X X X D-MX X X X X X X X X E-F X X X X X X E-F (LA#) X X X X X E-CO X X X E-MX X X F-CO X X X X X X F-MX X X X X X X X CO-MX X X X X X X
Table 1. Most useful traits for distinguishing between each pair of
A B C D E F CO MX A 81%/77% 85% 81% 98%/95% 77%/75% 88% 91% B 100% 97% 100% 84% 100% 100% C 83%/79% 78%/67% 91% 100% 100% D 100% 91%/88% 97% 92% E 100%/93% 100% 100% F 100% 100% CO 100% MX
A B C D E F CO MX A 66%/75% 77% 81% 99%/97% 76%/75% 88% 95% B 100% 98% 100% 94% 100% 100% C 61%/59% 84%/67% 94% 100% 100% D 100% 90%/88% 98% 91% E 100%/94% 100% 100% F 100% 100% CO 100% MX
Table 2. Shows the percentage of correctly classified specimen for each pairwise comparison of the clades using discriminant function analysis (DFA), jackknife analysis was use to verify the classifications and the resulting scores are shown in the tables above. In both tables the score from the dataset including lamellae counts are presented only when they improved the accuracy of the classification in which case they are the first of the two scores presented for that comparison.
Table 2a. (Top), shows the weighted classification score for each comparison. Highlighted cells represent Discriminant functions that had insignificant p=values for the Wilk’s lamba test (p ≥0.050) prior to the reverse stepwise DFA. The yellow cells represent comparisons which had Discriminant functions that were nearly significant (p=0.051-0.060) prior to the reverse stepwise DFA. Orange cells represent comparisons whose Discriminant functions were significant following the reverse stepwise DFA and red cells represent comparisons that the Discriminant function remained insignificant.
Table 2b. (Bottom), shows the unweighted classification score for each comparison. Dark green cells represent comparisons which resulted in ≥95% accuracy in correctly classifying the specimen. Light green represents 85%-95% accuracy in correct identifications. Light blue represents 80%-85% accuracy in correct identifications. Grey represents < 80% accuracy in correct identifications. 27
Discussion
Cryptic diversity
Using the Automatic Barcode Gap Discovery program (ABGD: Puillandre et al.,
2011) we identified 24 groups that represent potential species. Of those, eight groups represent the currently described species while the remaining 16 represent lineages corresponding to hypothetical species awaiting further systematic study (In progress).
This represents a potential for a 300% increase in species richness for this genus.
However, more extensive sampling throughout the range of each species is likely to reveal even higher levels of cryptic diversity. Recent studies have revealed similarly high amounts of cryptic diversity among reptiles on Caribbean islands (Hedges & Conn,
2012; Thomas & Hedges, 2007). In addition, geckos tend to contain greater numbers of cryptic species due to the tendency for closely related species to share a highly conserved morphology (Gamble et al., 2008; Rato & Harris, 2008; Perera & Harris,
2010).
This study focuses primarily on the cryptic diversity found within the Jamaican
Croaking Gecko (Aristelliger praesignis praesignis) and extensive sampling efforts were made throughout its range in Jamaica (N= 60). Within this single subspecies 10 hypothetical species (9 new) were identified by ABGD. Of the ten groups identified, one of hypothetical species was represented by a single sequence from a specimen that was sympatric with specimens from its closest relative (Clade B), and was collapsed into that clade. Another hypothetical species was also identified based on a single sequence. This hypothetical species lacked a museum specimen for morphological comparisons and therefore it not considered further. Six of the remaining clades of A. p. 28 praesignis, occur in Jamaica, two clades occur along the southern coast and four clades occur along the northern coast. Cayman Brac and Little Cayman account for the two remaining clades, each island having its own unique clade. Specimens from the
Cayman Islands were not incorporated into the morphological analyses for this project.
However, morphological differences between A. p. praesignis from the Cayman Islands and Jamaica have been noted elsewhere (Grant, 1940; Crombie, 1999).
In addition to the mtDNA evidence, the Jamaican clades were supported by morphological differences, although these differences are subtle and generally require a suite of traits to distinguish between pairs of clades. Discriminant function analysis revealed significant (p=0.05) morphological differences between most of the pairs of clades compared (20/28), while two of the eight remaining pairs were nearly significant
(p=0.051–0.060). For all but three pairs of clades, it was at least possible to identify a few traits that differed significantly between the pairs of clades being compared. For most pairs of clades, the members of each respective clade were correctly classified with greater than 80% accuracy, with over half (17/28) of the pairs being compared assigned to their correct clade with 95% accuracy or better. Clade A tended to be more difficult to distinguish from the other clades; the comparisons between A and B, A and C and A and F resulted in the correct assignment of members to their clades with around
75% accuracy. Only clades C and D were relatively indistinguishable from each other morphologically.
However, based on the mtDNA evidence, clade C is the most divergent lineage within the north coast group, and can be separated with moderate to high accuracy from all the remaining clades within this group. In addition, these clades are geographically 29 much closer than Clade D is to Clade C. Clade C occurs at Morant Point, in St. Thomas
Parish, Jamaica which is at the eastern most tip of the island while Clade D is found 155 km away at Duncans, in Trelawny Parish. The current inability to distinguish between these two clades morphologically is most likely a result of low sample size (Clade C is represented by only three specimens), as well as the high level of within group variation within these clades. Further evidence supporting the recognition of these clades as undescribed cryptic species comes from the syntopy at Port Antonio (Portland Parish) of two clades, Clade B and Clade F, which are members of the southern and northern coast groups, respectively. In addition, two closely related clades, E and F, are nearly sympatric at Port Maria/ Isla Cabarita in St. Mary Parish, Jamaica which are only separated by 0.75 km.
Phylogenetic Relationships
We sought to not only provide insight into the undescribed cryptic diversity within the genus Aristelliger, but also to gain a better understanding of the relationships of the previously described species within this genus. Prior to this study no comprehensive phylogenetic hypothesis had been proposed (Hedges, 1996), though a few predictions have been made (Hecht, 1951; Schwartz & Crombie, 1975). The results from the mtDNA phylogeny presented in this study supports some of these predictions, however, it also revealed some unexpected findings. The two subgenera form two highly divergent, well supported monophyletic clades. Within the subgenus of small species,
Aristelligella, there is evidence that the currently recognized species Aristelliger expectatus is a complex of species. For example, A. barbouri belongs to a different lineage of the A. expectatus complex than the two remaining species. Aristelliger 30
cochranae and A. reyesi form a clade along with a specimen from Grand Cayemite,
Haiti that is closest to another lineage of A. expectatus. The systematics of the
subgenus Aristelligella is currently under revision and the relationships within this subgenus will be discussed in detail in a later study (S. B. Hedges, unpublished).
Within the subgenus Aristelliger there are two major groups. One group contains
A. lar and A. hechti which form two well-defined monophyletic clades that are closest to
each other. Recently, it has been suggested that Aristelliger hechti occurs in northern
Hispaniola, but these populations have historically been misidentified as A. lar (Burns
et. al, 1992; Powell and Parmerlee, 1992 & Lever, 2003). The results of our analysis
confirm the existence of populations of A. hechti in Hispaniola. Representatives of what
was thought to be A. lar were taken from Altagracia which is in northeastern Dominican
Republic and from Barahona which is in southwestern Dominican Republic. The
specimen from northeastern Dominican Republic fell in the clade containing the A.
hechti specimen. This finding expands the range of A. hechti into Hispaniola from the
Caicos Islands and has important implications regarding potential errors in species
identification for this region.
The second major clade contains the remaining two species in this subgenus,
Aristelliger praesignis and A. georgeensis, the latter being a species known from coastal
islands off eastern Middle America from Mexico to Providencia and San Andres. Unlike
A. lar and A. hechti, these two species don’t form monophyletic closest relatives.
Instead, A. georgeensis is highly nested among populations currently recognized as A.
praesignis within Jamaica, being closest to populations of the North Coast group. The
North Coast group is diverse and contains four of the predicted species currently 31 recognized as A. praesignis, the clades associated with Little Cayman and Cayman
Brac, as well as A. georgeensis from Honduras (our only sample of that latter species).
The South coast contains the other two clades, currently recognized as A. praesignis.
Unlike A. praesignis from Little Cayman and Cayman Brac, the A. praesignis found on
Grand Cayman belong to Clade A, one of the two Jamaican south coast clades.
As previously mentioned, Aristelliger georgeensis was found to be tightly nested between the clades E and F from northwest Jamaica. In fact, ABGD supported A. georgeensis being a different species exclusive of Jamaica only in the recursive partition. While this finding is surprising, Aristelliger georgeensis has had a long history of being difficult to place taxonomically. In 1885, Boulenger suggested that A. georgeensis was not sufficiently different from A. praesignis and synonymized the two.
Morphology
While many species of geckos are difficult to distinguish morphologically and tend to harbor many cryptic species because of the tendency for geckos to retain ancestral traits (Gamble et al., 2008; Rato & Harris, 2008; Perera & Harris, 2010), the genus Aristelliger has stood out as being particularly enigmatic morphologically (Russell
& Bauer, 1993a; Hedges, 1996). This has led to taxonomic instability within the group and a great deal of frustration. Even 40 years ago it was thought that there were more species of Aristelliger than had been described, although no morphological traits could be found to separate them (Schwartz & Crombie, 1975; Crombie, 1999).
However, by integrating the knowledge gained from both molecular and morphological data it is possible to identify traits that may help in distinguishing between what Crombie (1999) considered to be “unquestionably different species.” Using a priori 32
knowledge based on mtDNA clade membership to assign specimen to groups, subtle
but significant morphological differences between the groups are revealed. Many of
these groups can be separated from each other with moderate to high accuracy. On
average, most groups are able to be separated from each other using an average of five
out of the 13 traits measured. A few groups are classified correctly 100% of the time
based on just two traits while at the other extreme some require up to 8 traits to
separate them. The most frequently used trait to distinguish between the groups was
eye diameter (ED). However, the inter-naris (IN) distance, height of the rostral scale
(RH), the height of the suture between the rostral scale and the first labial scale (SH),
mental width (MW), Eye Naris distance (EyN), Ear Naris distance (EN) and the Eye to
Ear distance (EE) were useful in distinguishing between at least half the clades each.
The least useful traits overall were the length of the ear and lamellae counts. However,
lamellae counts were the most important trait in separating clades C and E.
In some of the clades a single or a few traits stood out in their power to
distinguish between that clade and all or most of the remaining clades. The most
notable example of this is Mental Width in Clade B. While alone this trait isn’t sufficient
to distinguish between the clades, it was included in the suite of traits separating Clade
B from each of the other clades. The eye-naris distance (EyN) was useful in separating
Clade A from each of the clades with the exception of Clade B. Eye diameter was the most important trait in separating clade C from all other clades with the exception of
Clade-CO (Aristelliger georgeensis from Colombia) from which it could be classified with
100% accuracy using only the width of the 4th toe pad and inter-nares distance. Clade F
had multiple traits that were highly useful in separate it from the other clades. Rostral 33
Height and eye diameter were useful in separating Clade F from all other clades except for Clade-CO, while Ear Naris was useful in separating F from all clades except for B.
The primary objective of the morphological analyses was to provide preliminary evidence that the hypothetical species revealed in the mtDNA analyses were morphologically different. The formal descriptions of those species will be presented elsewhere. Preliminary morphological evaluation of two populations of Aristelliger georgeensis was undertaken due to the surprising finding that A. georgeensis was nested so tightly within the A. p. praesignis species complex and the even more surprising finding that it had the least support for being a distinct clade, despite having morphological differences established in the literature. These two species differ in maximum size (A. georgeensis, SVL=115 mm (Bauer & Russell, 1993); A. praesignis,
SVL=95 mm (this study: Clade B at Port Antonio)), as well as color pattern. However, populations of A. georgeensis from Honduras, where our genetic samples came from, are smaller with the maximum SVL of 95 mm (James R. McCranie, personal communication).
Based on morphological data from a population of A. georgeensis from Quintana
Roo, Mexico, and a population from San Andreas Island, Columbia, we found evidence that not only is A. georgeensis morphologically different from A. praesignis, they are also morphologically distinct from each other. However, further morphological data, as well as molecular data (if possible) are needed from this group. It is likely that A. georgeensis also forms a complex of cryptic species.
34
Biogeography:
The closest living relative of the genus Aristelliger is the genus Quedenfeldia
(Moroccan day geckos: Gamble et al., 2008), which is a North African endemic genus comprised of only 2 species (Barata, 2012). These two genera split approximately 70
Ma (Gamble et al., 2008), following the opening of the Atlantic Ocean. It is likely that the lineage giving rise to the genus Aristelliger evolved on Africa before dispersing to the
Caribbean between 37.2 Ma when the islands became permanently subaerial (Iturralde-
Vinent & MacPhee, 1999), and 22.5 Ma when the two subgenera spit. The center of origin for this genus is probably Hispaniola as this island has representatives of both subgenera, and the Dominican Republic was one of the first landmasses to become subaerial (Donnelly, 1992). The earlier divergence time of Aristelligella and the similarity of body size between the subgenus Aristelligella and Quedenfedia, suggest that the larger body size of the subgenus Aristelliger is the derived condition.
Shortly after the emergence of Jamaica 10 Ma (Donovan 2002; Mitchell 2004;
Donovan & Paul, 2011), a population of the larger subgenus, Aristelliger, colonized the island—likely arriving via flotsam carried by the currents from Hispaniola. Once in
Jamaica the population was divided into northern and southern clades reflecting the geography of the island. The northern coast is higher than the southern coast, with the center of the island consisting mainly of a limestone plateau, which is between about
600 and 750 meters high in most places (Donovan 2002; Mitchell 2004; Donovan &
Paul, 2011). Because Aristelliger praesignis prefers low lying coastal areas this served as a barrier to gene flow between the northern and southern coasts. 35
Clade C, from Morant Point at the eastern tip of Jamaica was the first of North
Coast clades to diverge. It is likely that they became isolated by the formation of the
Blue and John Crow Mountains, somewhere between 5–10 Ma (Comer, 1974; Hedges,
2001) , which is supported by the date of divergence from this study (6.7 Ma). These mountain ranges also likely serve as the eastern boundary between the Northern and
Southern clades. The northern coast is more genetically diverse than the southern coast. This is likely due to the more fragmentary nature of the coastal plain, their preferred habitat, isolated by mountains and steep cliffs that are found on the northern coast. Clades E and F are isolated from each other by the Blue Mountain range. Clades
D and E are likely separated by an extension of the Dry Harbor mountains. To the west the boundary between the northern and southern clades likely occurs at Montego Bay which is bordered by mountainous terrain. The southern coast has much more extensive coastal plains. It is likely that the barrier to dispersal between clades A and B is Portland Blight, which would have extended much further inland during the Pliocene when these two clades diverged, along with most of the other clades. However, the clade containing E, and F+ Aristelliger georgeensis is much younger, diverging only recently, during the Pleistocene.
There have been three dispersals out of Jamaica; one was dispersal from the southwestern coast to Grand Cayman. This was probably a recent dispersal since there is essentially no phylogenetic difference between the specimen from Grand Cayman and the Jamaican Clade A specimen. The clades on Little Cayman and Cayman Brac were due to a single dispersal from the northeast coast of Jamaica via the Rio Grande to one of the islands followed by a subsequent dispersal from that island to the other 36
island during the Pliocene. During the Pleistocene, another dispersal event, also via the
Rio Grande River this time to Central America, gave rise to Aristelliger georgeensis.
Summary:
The taxonomic relationships of the species within the genus Aristelliger remained
enigmatic prior to this study due the retention of ancestreal morphology as well as high
levels of intraspecific variation within traits that are traditionally used in gecko
systematics (Crombie, 1999). However, as with other groups whose relationships have
been difficult to resolve for these reasons, the use of molecular data has proven
valuable in clarifying these relationships (Carranza & Arnold 2006). The results from the
molecular analyses allowed us to address numerous questions that have been raised
concerning members of the genus. For example we were able to confirm the presence
of Aristelliger hechti in Hispaniola. In addition we were able to identify potential cryptic
species, which if supported by morphological data have the potential to triple the
species diversity within the genus.
We also found that Aristelliger praesignis forms a complex of species with A.
georgeensis nested within it. The hypothetical species identified by the molecular data were morphologically distinguishable with high accuracy based on suites 2—9 traits.
This evidence as well as the syntopy of two of these hypothetical species, and near
sympatry of two others lends strong support to the validity of these as unique species.
These findings are significant because it reveals that the genus Aristelliger is
considerably more diverse than was previously thought. Because the newly defined
species have much smaller distributions, these results also have a bearing on their
conservation status. 37
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