Research Article |
Corresponding author: Ingi Agnarsson ( iagnarsson@gmail.com ) Academic editor: Miquel Arnedo
© 2016 Ingi Agnarsson, Stephanie M. LeQuier, Matjaž Kuntner, Ren-Chung Cheng, Jonathan A. Coddington, Greta Binford.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Agnarsson I, LeQuier SM, Kuntner M, Cheng R-C, Coddington JA, Binford G (2016) Phylogeography of a good Caribbean disperser: Argiope argentata (Araneae, Araneidae) and a new ‘cryptic’ species from Cuba. ZooKeys 625: 25-44. https://doi.org/10.3897/zookeys.625.8729
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The Caribbean islands harbor rich biodiversity with high levels of single island endemism. Stretches of ocean between islands represent significant barriers to gene-flow. Yet some native species are widespread, indicating dispersal across oceans, even in wingless organisms like spiders. Argiope argentata (Fabricius, 1775) is a large, charismatic, and widespread species of orb-weaving spider ranging from the United States to Argentina and is well known to balloon. Here we explore the phylogeography of A. argentata in the Caribbean as a part of the multi-lineage CarBio project, through mtDNA haplotype and multi-locus phylogenetic analyses. The history of the Argiope argentata lineage in the Caribbean goes back 3-5 million years and is characterized by multiple dispersal events and isolation-by-distance. We find a highly genetically distinct lineage on Cuba which we describe as Argiope butchko sp. n. While the argentata lineage seems to readily balloon shorter distances, stretches of ocean still act as filters for among-island gene-flow as evidenced by distinct haplotypes on the more isolated islands, high FST values, and strong correlation between intraspecific (but not interspecific) genetic and geographic distances. The new species described here is clearly genetically diagnosable, but morphologically cryptic, at least with reference to the genitalia that typically diagnose spider species. Our results are consistent with the intermediate dispersal model suggesting that good dispersers, such as our study species, limit the effect of oceanic barriers and thus diversification and endemism.
Biogeography, CarBio, dispersal, diversification, GAARlandia, Intermediate dispersal model, Isolation by distance model
The Caribbean diversity hotspot has been colonized by a number of lineages via varying routes over millions of years. As is typical of other old oceanic islands, the archipelago’s isolation helped form numerous single-island endemic species (
Being wingless, a relatively small proportion of arachnid lineages tend to colonize ocean islands. Single-island endemism is common in successfully colonizing lineages (
Species of the genus Argiope are large, sexually dimorphic, charismatic spiders with brightly colored abdomens (
Here, we present mtDNA and morphological data on Argiope argentata collected throughout the Caribbean to reveal phylogeographical patterns within the Caribbean, to test the degree of genetic structure within and among islands, and to measure divergence in cases where genetic patterns reflect geography. We verify relationships among species with a multi-locus phylogenetic approach, and we also describe a new species, Argiope butchko sp. n., previously thought to represent Cuban populations of A. argentata.
Specimens of Argiope argentata s. l. were collected diurnally using standard aerial searching and beating methods from 2011-2015 across the Caribbean and in SE USA (Fig.
A dated phylogeny of Argiope argentata in the Caribbean, and other Argiope relatives. Shown are the results of tree based species delimitation analyses (GMYC method) on a BEAST phylogeny (node ages in million years) and the location of spiders used in this study (inset picture). Asterisk denotes posterior probability support >95%. The OTUs (operational taxonomic units) correspond to a cryptic species, Argiope butchko sp. n., from Cuba (argentataCU) and populations from other Caribbean islands (argentataCAR) plus mainland (argentataUS) treated as conspecific (A. argentata).
DNA was isolated from 85 A. argentata s.l. and 13 other Argiope species with the QIAGEN DNeasy Tissue Kit (Qiagen, Inc., Valencia, CA), or using phenol extraction (Suppl. material
For Bayesian analyses, the GTR+I+G model was selected as the appropriate substitution model by the AIC criterion (
To test the phylogenetic relationships from COI data, we also ran Bayesian analysis with a multi-locus dataset with two nuclear markers (28S and Histone 3) and 1–2 exemplars per species. The PCR reactions of 28S and Histone 3 followed established protocols for argiopine spiders (
To test for cryptic species in A. argentata, we used a combination of tree-based species delimitation methods and genetic distances. For tree-based species delimitation method, the General Mixed Yule-Coalescent model with single threshold (GMYC) (
Descriptive statistics for K2P (Kimura 2-parameter) distances within and between the molecular operational taxonomic units (OTUs), which were identified by molecular species delimitation methods.
Within OTUs | |||
---|---|---|---|
OTU | N | K2P | |
Mean | Std. Err | ||
argentataCAR | 74 | 0.009 | 0.002 |
argentataUS | 3 | 0.018 | 0.004 |
argentataCU | 10 | 0.006 | 0.001 |
Between OUTs | |||
OTU 1 | OTU 2 | K2P | |
Mean | Std. Err | ||
argentataCAR | argentataUS | 0.029 | 0.006 |
argentataCAR | argentataCU | 0.061 | 0.010 |
argentataUS | argentataCU | 0.064 | 0.010 |
Fst and Kxy indexes were calculated in DNAsp v5 (
Haplotype networks were constructed using median-joining networks (
Adult males and females were imaged using a Visionary Digital BK Plus digital imaging system. Specimens arranged in hand sanitizer and covered in 95% ethanol were photographed at dorsal, ventral, and lateral angles. Taxonomic measurements were derived from photographs in Adobe Photoshop. Genitalia observations and illustrations were made from photographs and by dissecting out the epigyna, digested in potassium hydroxide solution to remove soft tissue to make internal structures visible.
A fragment of COI (659 bp) was obtained for all individuals, and with added data from Genbank, making up a total of 107 sequences, including outgroups and 87 individuals morphologically identified as A. argentata. 540 base pairs overlapped for all individuals and missing data was 5.1%. Bayesian analyses of this dataset produced a topology that, with some internal node exceptions, was well supported (Figs
The GMYC analysis split the COI data into 11 OTUs, including eight correctly identified outgroup species. GMYC model provided a significantly better fit to the data than the null hypothesis of no structure (likelihood ratio: 27.06, P < 0.001), thus identifying 3 OTUs within A. argentata s. l. (Fig.
Other measures of nucleotide differences (Kxy) and gene flow (FST) likewise indicate particularly high distinction and genetic isolation of these lineages (Suppl. material
Regression analysis between geographic and genetic distances among all specimens of A. argiope s.l. included here. Blue dots and line represent within species comparisons and red dots and line those among the two species as here defined. Black line is regression across all data. Geographical distances well explain genetic distances within species, but not between the species, as expected.
Finally, haplotype analyses indicate clear phylogeographic structuring of COI haplotypes despite generally shallow divergences among islands (Fig.
A haplotype network of Caribbean A. argentata and A. butchko sp. n. Haplotypes are colored by locality as indicated, circle size reflects number of individuals carrying that haplotype from 1-7 in total, open circles represent unobserved haplotypes. Hash marks indicate mutational differences among haplotypes. Inset photograph is of a female A. butchko.
Archipelagos such as the Caribbean provide opportunities for colonization followed by isolation, restriction of gene flow, and the formation of local endemics (Ricklefs and Birmingham 2008). However, the degree of endemism will depend, in part, on the dispersal ability of the lineages in question (e.g.
We find strong evidence for two species in our dataset, the widespread A. argentata and one new species, A. butchko sp. n. from Cuba. Both genetic distances (Table
Based on this finding we examined in detail the comparative morphology of A. argentata and the putative new species from Cuba, here described as A. butchko sp. n. We found no diagnostic differences in the morphology of male and female genitalia that would be consistent with the deep genetic divergence (~6.3% average sequence divergence, Table
We note that our main results are based on a single mitochondrial locus and thus our phylogeographic conclusions are restricted to the picture expected from female inheritance. Nevertheless, multi-locus phylogenetic analyses support the general conclusions.
Female holotype Argiope butchko sp. n. a dorsal b lateral c ventral; Male paratype Argiope butchko sp. n. d dorsal e lateral f ventral g external epigynum h external epigynum illustration showing spermatheca and spiraling ducts i internal epigynum illustration dorsal m palp lateral n palp ventral; Argiope argentata j external epigynum k external epigynum illustration showing spermatheca and spiraling ducts l internal epigynum illustration dorsal o palp lateral p palp ventral.
Comparative morphology of the male palpal organ of the widespread A. argentata and the new A. butchko. No clearly diagnostic features were identified in the new species, though slight differences in the terminal parts of the median apophysis and the embolus are observed and merit further comparative investigation.
Consistent with predictions of the intermediate dispersal model, our analyses of Argiope argentata mtDNA haplotype diversity and phylogeography across the Caribbean established that geographical structuring of haplotypes of this good disperser taxon was mostly consistent with a single widespread species model. Nevertheless, specimens from Cuba were deeply divergent from the remaining areas in all analyses, which we take as evidence for discovery of a cryptic species here described as A. butchko sp. n. ‘Hidden’ taxonomic diversity in the Caribbean is being revealed in multiple lineages by the CarBio project (
The species epithet, a noun in apposition, honors the memory of Dennis Butchko, an inspiring science teacher.
Female holotype from Siboney, Santiago de Cuba (19.9608°N, 75.7076°W), April 1, 2012, Col. Team CarBio, deposited in the Smithsonian (
Argiope butchko sp. n. differs from all other Argiope except A. argentata by the presence of the embolic distal curl (Levi, 2004: fig. 43, arrow). No distinct feature of the male palp and female epigynum were found that reliably diagnose A. butchko sp. n. from A. argentata.
Argiope butchko sp. n. and A. argentata can be diagnosed from one another, and other related Argiope species, on the basis of the following unique, synapomorphic, mtDNA nucleotide substitutions at the following standard DNA barcode alignment positions in each species (following
A. butchko: A (127), C (133), C (157) C (178), T (190), G (208), C (226), A (293), G (316), A (379, G (502), G (508), C (607); A. argentata: G (49), G (211), A (508), A (511), G (643).
Males and females of this species closely resemble Argiope argentata (
Dimensions (mm). Holotype (female) - Total body length excluding chelicera 10.74, carapace length 5.00, carapace width 4.36. Leg I: femur length 8.28, patella and tibia length 8.63mm, metatarsus 8.00, tarsus 2.09. Leg II: patella and tibia length 8.06. Leg III: patella and tibia length 4.66. Leg IV: patella and tibia length 7.13.
Variation (mm). Female (N=4) - Total body length ranged from 10.29–10.74, carapace length 3.84–5.00, carapace width 3.16–4.36. Leg I: femur length 6.50–8.28, patella and tibia length 6.40–8.63, metatarsus 5.77–8.00, tarsus 1.80–2.09. Leg II: patella and tibia length 7.83–8.06. Leg III: patella and tibia length 3.73–4.66. Leg IV: patella and tibia length 4.66–7.13. Male (N=3) - Total body length ranged from 2.88–3.44, carapace length 1.70–1.88, carapace width 1.44–1.57. Leg I: femur length 1.62–2.14, patella and tibia length 1.99–2.37, metatarsus 1.74–1.88, tarsus 0.81–0.88. Leg II: patella and tibia length 1.41–1.90. Leg III: patella and tibia length N/A. Leg IV: patella and tibia length 1.40–1.63.
The species is restricted to Cuba.
Three embolus tips were found embedded in the epigynum of a female A. butchko, one in the left opening and two in the right opening (Fig.
Many thanks to all the members of team CarBio and our collaborators for tireless effort in the field over the last four years. Special thanks to collaborators in Cuba (Alexander Sanchez, Giraldo Alayon, Franklyn Cala-Riquelme and others) and Hispaniola (Solanlly Carrero Jiménez, Gabriel de los Santos) for invaluable help with organization and execution of logistically complex expeditions and ongoing collaboration. We thank Matjaž Gregorič, Klemen Čandek, and Gabriele Uhl for kind help with obtaining additional samples. Funding for this work came from NSF DEB-1050187-1050253-1314749 to IA and GB, and the Smithsonian Institution 2013 SI Barcode Network to JC and IA. MK and RCC were supported by the Slovenian Research Agency (grants P1-10236 and J1-6729 to MK).
Figure S1
Data type: phylogeny data
Explanation note: Bayesian phylogeny based on analysis in MrBayes using CO1 data
Figure S2
Data type: phylogeny data
Explanation note: Bayesian phylogeny based on analysis in MrBayes using the two nuclear genes.
Figure S3
Data type: phylogeny data
Explanation note: Detailed results from the BEAST analysis summarized in Fig.
Table S1
Data type: specimen data
Explanation note: Specimen data including the Genbank accession number.
Table S2
Data type: specimen data
Explanation note: Results of Fst and Kxy (average number of nucleotide differences) analyses among specimens from all islands and mainland. The Cuban specimens, here described as a new species, stand out in both analyses indicating genetic isolation and divergence.
Table S3
Data type: specimen data
Explanation note: Genetic and Geographic distances among all ingroup specimens used in generating Figure