Phylogeography of the New World Sphingonotus species

The inferred phylogeny interpreted against the background of contemporary species distributions lets us argue that (i) grasshoppers of the tribe Sphingonotini are mainly distributed in the Old World. However, (ii) the focal species found in the Neotropics, i.e. on the Galapagos Islands and in the Caribbean, belong to the Sphingonotini rather than to the Trimerotropini, which is the predominant tribe in the New World. Hence, our analyses reject our first hypothesis that the taxonomic assignment of the Caribbean and Galapagos species to the tribe Sphingonotini is wrong. Rather our data support the hypothesis that the Caribbean (i.e. Atlantic) and the Galapagos Archipelago (i.e. Pacific) species are members of the Sphingonotini.

It has been suggested that the occurrence of Sphingonotus on Galapagos might be the result of a recent introduction from Europe [16]. This hypothesis can be rejected as well, since the species represent rather old lineages within the genus and are much older than most Old World species and diverged prior to any potential introduction date. While the dating is very crude the resulting age estimates are more likely an underestimate than an overestimate; the divergence between the two major clades (Trimerotropini from the New World and Sphingonotini from the Old World) was here estimated at approximately 24.4 million years ago. This dating estimate is more recent (yet both estimates have overlapping 95% HPD) than the estimate derived from a more comprehensive study which dated the split between the clades at about 35 mya [23]. The same split was dated even further back (~55 mya) by a study by Chapco & Contreras [34]. The estimate derived here is therefore a minimum estimate of the age with the lineages likely being much older. The ages of the Galapagos endemics with more than 7 mya at the basis of the lineages predate the origin of the islands.

The observed relationships may be explained by long-distance dispersal via the mainland leading to the colonization of the islands with subsequent extinction on the mainland. One might even speculate that the Sphingonotini might have colonized the American continent (e.g. [23]) and later been displaced by Trimerotropini, except for the oceanic island populations. This is supported by the high age of the islands endemics predating the ages of the islands. Alternatively, the New World Sphingonotus species might have reached the islands via rare long-distance, trans-Atlantic dispersal events. The first colonization step was then likely to the Caribbean, which is supported by the phylogeny. A reasonable number of studies have shown trans-Atlantic dispersal of a variety of animal and plant taxa [35–38]. For example, a study by Carranza and colleagues [39] showed a case of long-distance dispersal, where Tarentola Geckos invaded the Caribbean from Africa [39]; South America has been colonized by Hemidactylus Geckos from Africa [40], and the Americas were colonized from Africa by the grasshopper genus Schistocerca [36].

The Galapagos lineages of Sphingonotus appear to be older than many of the islands and hence a previous mainland distribution with subsequent extinction appears more likely. A continental extinction of the genus would also explain the lack of monophyly of the New World Sphingonotini. However, with our data we are not able to support with confidence either of the following hypotheses: (1) the Sphingonotini had a wider New World distribution which has been largely replaced by the Trimerotropini except for relict occurrences of Sphingonotus on the archipelagos or (ii) the Sphingonotini of the Galapagos archipelago and Hispaniola are the result of trans-Atlantic colonization.

Island colonization and differentiation

In the past, Sphingonotus fuscoirroratus from Galapagos had been divided into two species with several subspecies [28]. Subsequently, these taxa were synonymised as only limited morphological variation between island lineages was found [26]. Our analyses suggest that each island indeed has its own distinct genetic lineage which supports the original species or subspecies status. The extent of genetic divergence of the island populations suggests that no or very little gene flow between islands exists.

Generally, inter-island radiations are typical for the Galapagos as a result of the large distance to the mainland and the relatively high distances between most islands. This can partly be confirmed here (at least for four islands). Similar radiations on the Galapagos are known for mockingbirds (Nesomimus) [10], tenebrionid beetles [41], iguanas (Conolophus) [42], and the Galapagos lava lizards [43]. The lack of monophyly of S. fuscoirroratus due to the position of the San Cristobal lineage might be caused by insufficient resolution of the data or by extinction of true sister species on the American continent. However, another explanation might be that this island was colonized independently from the others as has been shown for the Canary Islands as well [22]. However, this hypothesis would require the assumption that both lineages converged substantially in morphology when adapting to the island habitats.

Conclusion

Our analyses support that the Galapagos endemic S. fuscoirroratus and the Caribbean endemic S. haitensis indeed belong to the tribe Sphingonotini and we therefore reject the hypothesis that these species had been wrongly assigned to the Sphingonotini. The colonization is rather ancient which allows us to reject the hypothesis that the studied species were the result of anthropogenic translocation. However, we cannot infer with certainty if the populations are relicts of a previously more widespread distribution or the result of long-distance, trans-Atlantic dispersal. In demonstrating a close phylogenetic relationship of Galapagos endemic species to Old World taxa, this study highlights the need to include geographically distantly distributed taxa in phylogeographic studies. Following the deep genetic splits detectable for our samples from Galapagos Islands, we assume that at least three to four distinct Sphingonotus species exist on the archipelago. It is likely that further genetic lineages are present on other islands that had not been studied here in concert with the original designation as species and subspecies [28].

Study species

Grasshoppers of the genus Sphingonotus are widely distributed across major parts of the Palaearctic and Palaeotropic regions. A supposedly close relative, the genus Trimerotropis, can be found exclusively in the Nearctic and Neotropic region [16, 17]. The genera Trimerotropis and Sphingonotus show strong morphological similarities; however, representatives of Trimerotropis are mostly larger [16, 24]. Both genera had been grouped in the tribe Sphingonotini for many decades, but recently the genus Trimerotropis was re-assigned to the previously erected Trimerotropini [23, 44]. Both genera are species-rich with 142 species for Sphingonotus and 52 Species for Trimerotropis [17].

Sampling

In total, 104 individuals belonging to 44 species from four continents were included in the analyses (Table 1). Specimens were collected by hand or netted and subsequently frozen or stored in ethanol. Many samples were obtained from museums or colleagues. None of the collected species are protected and no sampling was performed on protected land aside from the Galapagos. Sampling activities on Galapagos were performed by D. Otte (ANSP, Philadelphia) and S. B. Peck (Carleton University, Ottawa, Canada) under permission of the Galapagos National Park (F. Cepeda, A. Izurieta and E. Cruz, Superintendents, Department of Forestry, Ministry of Agriculture, Republic of Ecuador). The Gomphocerinae Cibolacris parviceps and the Oedipodinae Chortophaga viridifasciata served as outgroups in all analyses. Details about all individuals collected and used for this study are given in Table 1.

Molecular analyses

Genomic DNA was extracted from dried or ethanol preserved hind leg muscle tissue using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Inc., Valencia, CA) following the manufacturer’s protocol for tissue samples. We amplified two mitochondrial and one nuclear gene fragment using a standard PCR protocol. Primers for the mitochondrial NADH Dehydrogenase subunit 5 (ND5) were obtained from Su and colleagues [45] and for COI from Husemann and colleagues [23]. The primers for Histone 3 (H3) were taken from Colgan and colleagues [46]. PCR reactions were performed using the following setup: 36.6 μl of diH₂O, 6 μl of 10 x PCR buffer (reaction concentration 1x), 4.8 μl of dNTP mixture (0.2 μM each), 0.6 μl of DyNAzyme DNA Polymerase (1.2 U, Finnzymes, USA), 3 μl of each primer (0.5 μM, Integrated DNA technologies, USA) and 6 μl of DNA template adding up to a total volume of 60 μl. Amplification conditions were as follows: 94°C for 3 min, followed by 30 cycles of 94°C for 1 min denaturation, 48–57°C 1 min annealing and 72°C for 2 min elongation, with a final elongation step at 72°C for 10 min.

PCR products were visualized on a 1% agarose gel stained with Gel Red (0.1x, Biotium, USA and purified using Solid-phase Reversible Immobilization (SPRI) [47] with carboxylated magnetic beads (Bangs Laboratories, USA) and a 96-Ring SPRIplate (Agencourt, USA). The purified PCR products were sequenced at the Yale Sequencing Facility (New Haven, CT, USA). All sequences were deposited in Genbank; accession numbers are given in Table 1.

Phylogenetic analyses

Sequences were inspected, trimmed and aligned using the MAFFT algorithm in Geneious 5.0.3 [48]. Further we used sequences from previous studies [18–20, 22, 23]. All genes were subsequently analyzed as combined data set. In a first step we identified the best partitioning scheme treating codon positions separately and determined the most suitable substitution models using PartitionFinder v.1.1.1 [49]. We performed two runs of PartitionFinder, one including the models implemented in MrBayes and one including the models implemented in BEAST. We then analyzed the concatenated partitioned data set with MrBayes v.3.1.2 [50]. We ran MrBayes for 50 million generations sampling every 5000 generations. A burn-in of 25% of trees was discarded before constructing a consensus tree. In addition we used BEAST v. 1.8.0 [51] to analyze the data in a supertree framework. The input file for BEAST was setup with BEAUti v. 1.8.0 (implemented in the BEAST package). We used the partitioning scheme from PartitionFinder to link the substitution models. The clock models were linked for mitochondrial genes. The trees were linked for all data. We used the Yule prior as recommended for analyses at species and genus levels and ran the analyses for 100 million iterations sampling every 10,000 iterations. The log-files were checked in Tracer v.1.5 [52] to check for convergence. A burn-in of 1000 trees was discarded before generating a consensus tree. All trees were visualized using FigTree v.1.3.1 [53].

In addition we obtained coarse estimates of divergence dates by applying a molecular clock approach. We used published substitution rates of 0.0113 for ND5 [23] and 0.01 for COI estimating the rate for H3 and applied a strict clock in BEAST v.1.8.0 [51]. No better calibration was possible as no suitable fossil data is available and using island ages as calibration points appeared inappropriate considering that we intended to estimate the divergence times of island lineages. The analysis was run for 100 million generations sampling every 10,000 generations. Trees were summarized with TreeAnnotator and visualized with FigTree.

In a last step we obtained evidence for the origin of the Galapagos taxa by using statistical DIVA and Bayes-Lagrange analyses as implemented in RASP v.3.0 [54]. We used the trees generated by our BEAST run as input and defined the geographic areas as follows: A—N America, B—Africa (including Cape Verde), C—Europe (including the Canary Islands), D—Galapagos Islands, E—Caribbean, F—Asia, G—S America. The maximum areas per node were set as 2.