Regina L. Cunha

Neogastropod phylogenetic relationships based on entire mitochondrial genomes

BackgroundThe Neogastropoda is a highly diversified group of predatory marine snails (Gastropoda: Caenogastropoda). Traditionally, its monophyly has been widely accepted based on several morphological synapomorphies mostly related with the digestive system. However, recent molecular phylogenetic studies challenged the monophyly of Neogastropoda due to the inclusion of representatives of other caenogastropod lineages (e.g. Littorinimorpha) within the group. Neogastropoda has been classified into up to six superfamilies including Buccinoidea, Muricoidea, Olivoidea, Pseudolivoidea, Conoidea, and Cancellarioidea. Phylogenetic relationships among neogastropod superfamilies remain unresolved.ResultsThe complete mitochondrial (mt) genomes of seven Neogastropoda (Bolinus brandaris, Cancellaria cancellata, Conus borgesi, Cymbium olla, Fusiturris similis, Nassarius reticulatus, and Terebra dimidiata) and of the tonnoidean Cymatium parthenopeum (Littorinimorpha), a putative sister group to Neogastropoda, were sequenced. In addition, the partial sequence of the mitochondrial genome of the calyptraeoidean Calyptraea chinensis (Littorinimorpha) was also determined. All sequenced neogastropod mt genomes shared a highly conserved gene order with only two instances of tRNA gene translocation. Phylogenetic relationships of Neogastropoda were inferred based on the 13 mt protein coding genes (both at the amino acid and nucleotide level) of all available caenogastropod mitochondrial genomes. Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses failed to recover the monophyly of Neogastropoda due to the inclusion of the tonnoidean Cymatium parthenopeum within the group. At the superfamily level, all phylogenetic analyses questioned the taxonomic validity of Muricoidea, whereas the monophyly of Conoidea was supported by most phylogenetic analyses, albeit weakly. All analyzed families were recovered as monophyletic except Turridae due to the inclusion of Terebridae. Further phylogenetic analyses based on either a four mt gene data set including two additional Littorinimorpha or combining mt and nuclear sequence data also rejected the monophyly of Neogastropoda but rendered rather unresolved topologies. The phylogenetic performance of each mt gene was evaluated under ML. The total number of resolved internal branches of the reference (whole-mt genome) topology was not recovered in any of the individual gene phylogenetic analysis. The cox2 gene recovered the highest number of congruent internal branches with the reference topology, whereas the combined tRNA genes, cox1, and atp8 showed the lowest phylogenetic performance.ConclusionPhylogenetic analyses based on complete mt genome data resolved a higher number of internal branches of the caenogastropod tree than individual mt genes. All performed phylogenetic analyses agreed in rejecting the monophyly of the Neogastropoda due to the inclusion of Littorinimorpha lineages within the group. This result challenges morphological evidence, and prompts for further re-evaluation of neogastropod morphological synapomorphies. The important increase in number of analyzed positions with respect to previous studies was not enough to achieve conclusive results regarding phylogenetic relationships within Neogastropoda. In this regard, sequencing of complete mtDNAs from all closely related caenogastropod lineages is needed. Nevertheless, the rapid radiation at the origin of Neogastropoda may not allow full resolution of this phylogeny based only on mt data, and in parallel more nuclear sequence data will also need to be incorporated into the phylogenetic analyses.

Phylogeny and Diversification Patterns among Vesicomyid Bivalves

Vesicomyid bivalves are among the most abundant and diverse symbiotic taxa in chemosynthetic-based ecosystems: more than 100 different vesicomyid species have been described so far. In the present study, we investigated the phylogenetic positioning of recently described vesicomyid species from the Gulf of Guinea and their western Atlantic and Pacific counterparts using mitochondrial DNA sequence data. The maximum-likelihood (ML) tree provided limited support for the recent taxonomic revision of vesicomyids based on morphological criteria; nevertheless, most of the newly sequenced specimens did not cluster with their morphological conspecifics. Moreover, the observed lack of geographic clustering suggests the occurrence of independent radiations followed by worldwide dispersal. Ancestral character state reconstruction showed a significant correlation between the characters “depth” and “habitat” and the reconstructed ML phylogeny suggesting possible recurrent events of ‘stepwise speciation’ from shallow to deep waters in different ocean basins. This is consistent with genus or species bathymetric segregation observed from recent taxonomic studies. Altogether, our results highlight the need for ongoing re-evaluation of the morphological characters used to identify vesicomyid bivalves.

High connectivity across the fragmented chemosynthetic ecosystems of the deep Atlantic Equatorial Belt: efficient dispersal mechanisms or questionable endemism?

Chemosynthetic ecosystems are distributed worldwide in fragmented habitats harbouring seemingly highly specialized communities. Yet, shared taxa have been reported from highly distant chemosynthetic communities. These habitats are distributed in distinct biogeographical regions, one of these being the so‐called Atlantic Equatorial Belt (AEB). Here, we combined genetic data (COI) from several taxa to assess the possible existence of cryptic or synonymous species and to detect the possible occurrence of contemporary gene flow among populations of chemosynthetic species located on both sides of the Atlantic. Several Evolutionary Significant Units (ESUs) of Alvinocarididae shrimp and Vesicomyidae bivalves were found to be shared across seeps of the AEB. Some were also common to hydrothermal vent communities of the Mid‐Atlantic Ridge (MAR), encompassing taxa morphologically described as distinct species or even genera. The hypothesis of current or very recent large‐scale gene flow among seeps and vents was supported by microsatellite analysis of the shrimp species Alvinocaris muricola/Alvinocaris markensis across the AEB and MAR. Two nonmutually exclusive hypotheses may explain these findings. The dispersion of larvae or adults following strong deep‐sea currents, possibly combined with biochemical cues influencing the duration of larval development and timing of metamorphosis, may result in large‐scale effective migration among distant spots scattered on the oceanic seafloor. Alternatively, these results may arise from the prevailing lack of knowledge on the ocean seabed, apart from emblematic ecosystems (chemosynthetic ecosystems, coral reefs or seamounts), where the widespread classification of endemism associated with many chemosynthetic taxa might hide wider distributions in overlooked parts of the deep sea.

Replaying the tape: recurring biogeographical patterns in Cape Verde Conus after 12 million years

Isolated oceanic islands are excellent natural laboratories to test the relative role of historical contingency and determinism in evolutionary diversification. Endemics of the marine venomous snail Conus in the Cape Verde archipelago were originated from at least two independent colonizations of ‘small’ and ‘large’ shelled species separated by 12 million years. In this study, we have reconstructed phylogenetic relationships within large‐shelled Conus (C. ateralbus, C. pseudonivifer, C. trochulus, and C. venulatus) based on mitochondrial cox1 and nad4 haplotype sequences. The reconstructed molecular phylogeny revealed three well‐supported and relatively divergent clades (A, B, and C) that do not correspond to current species classification based on shell colour and banding patterns. Clade A grouped specimens assigned either to C. pseudonivifer or C. trochulus, clade B is composed of specimens assigned to C. venulatus, and clade C comprises specimens assigned either to C. venulatus or C. ateralbus. Geometric morphometric analyses found significant differences between the radular teeth shape of C. pseudonivifer/C. trochulus and C. venulatus/C. ateralbus. In clades A and B, northwestern Boavista and Maio specimens cluster together to the exclusion of eastern Boavista samples. In Sal, populations form a monophyletic island assemblage (clade C). The large‐shelled Conus have remarkably replicated biogeographical patterns of diversification of small‐shelled Conus. Similar selective forces (i.e. nonplanktonic lecithotrophy with limited larval dispersal and allopatric diversification) together with repeated instances of low sea level stands during glacial maxima that allowed connection between islands, have overcome the effect of historical contingency, and explain the observed recurring biogeographical patterns.

Evolutionary Patterns in Pearl Oysters of the Genus Pinctada (Bivalvia: Pteriidae)

Pearl oysters belonging to the genus Pinctada (Bivalvia: Pteriidae) are widely distributed between the Indo-Pacific and western Atlantic. The existence of both widely distributed and more restricted species makes this group a suitable model to study diversification patterns and prevailing modes of speciation. Phylogenies of eight out of the 11 currently recognised Pinctada species using mitochondrial (cox1) and nuclear (18S rRNA) data yielded two monophyletic groups that correspond to shell size and presence/absence of hinge teeth. Character trace of these morphological characters onto the molecular phylogeny revealed a strong correlation. Pinctada margaritifera appears polyphyletic with specimens from Mauritius grouping in a different clade from others of the French Polynesia and Japan. Hence, P. margaritifera might represent a species complex, and specimens from Mauritius could represent a different species. Regarding the putative species complex Pinctada fucata/Pinctada martensii/Pinctada radiata/Pinctada imbricata, our molecular analyses question the taxonomic validity of the morphological characters used to discriminate P. fucata and P. martensii that exhibited the lowest genetic divergence and are most likely conspecific as they clustered together. P. radiata and P. imbricata were recovered as monophyletic. The absence of overlapping distributions between sister lineages and the observed isolation by distance suggests that allopatry is the prevailing speciation mode in Pinctada. Bayesian dating analysis indicated a Miocene origin for the genus, which is consistent with the fossil record. The northward movement of the Australian plate throughout the Miocene played an important role in the diversification process within Pinctada.

Wider sampling reveals a non-sister relationship for geographically contiguous lineages of a marine mussel

The accuracy of phylogenetic inference can be significantly improved by the addition of more taxa and by increasing the spatial coverage of sampling. In previous studies, the brown mussel Perna perna showed a sister–lineage relationship between eastern and western individuals contiguously distributed along the South African coastline. We used mitochondrial (COI) and nuclear (ITS) sequence data to further analyze phylogeographic patterns within P. perna. Significant expansion of the geographical coverage revealed an unexpected pattern. The western South African lineage shared the most recent common ancestor (MRCA) with specimens from Angola, Venezuela, and Namibia, whereas eastern South African specimens and Mozambique grouped together, indicating a non-sister relationship for the two South African lineages. Two plausible biogeographic scenarios to explain their origin were both supported by the hypotheses-testing analysis. One includes an Indo-Pacific origin for P. perna, dispersal into the Mediterranean and Atlantic through the Tethys seaway, followed by recent secondary contact after southward expansion of the western and eastern South African lineages. The other scenario (Out of South Africa) suggests an ancient vicariant divergence of the two lineages followed by their northward expansion. Nevertheless, the “Out of South Africa” hypothesis would require a more ancient divergence between the two lineages. Instead, our estimates indicated that they diverged very recently (310 kyr), providing a better support for an Indo-Pacific origin of the two South African lineages. The arrival of the MRCA of P. perna in Brazil was estimated at 10 [0–40] kyr. Thus, the hypothesis of a recent introduction in Brazil through hull fouling in wooden vessels involved in the transatlantic itineraries of the slave trade did not receive strong support, but given the range for this estimate, it could not be discarded. Wider geographic sampling of marine organisms shows that lineages with contiguous distributions need not share a common ancestry.

Ancient Divergence in the Trans-Oceanic Deep-Sea Shark Centroscymnus crepidater

Unravelling the genetic structure and phylogeographic patterns of deep-sea sharks is particularly challenging given the inherent difficulty in obtaining samples. The deep-sea shark Centroscymnus crepidater is a medium-sized benthopelagic species that exhibits a circumglobal distribution occurring both in the Atlantic and Indo-Pacific Oceans. Contrary to the wealth of phylogeographic studies focused on coastal sharks, the genetic structure of bathyal species remains largely unexplored. We used a fragment of the mitochondrial DNA control region, and microsatellite data, to examine genetic structure in C. crepidater collected from the Atlantic Ocean, Tasman Sea, and southern Pacific Ocean (Chatham Rise). Two deeply divergent (3.1%) mtDNA clades were recovered, with one clade including both Atlantic and Pacific specimens, and the other composed of Atlantic samples with a single specimen from the Pacific (Chatham Rise). Bayesian analyses estimated this splitting in the Miocene at about 15 million years ago. The ancestral C. crepidater lineage was probably widely distributed in the Atlantic and Indo-Pacific Oceans. The oceanic cooling observed during the Miocene due to an Antarctic glaciation and the Tethys closure caused changes in environmental conditions that presumably restricted gene flow between basins. Fluctuations in food resources in the Southern Ocean might have promoted the dispersal of C. crepidater throughout the northern Atlantic where habitat conditions were more suitable during the Miocene. The significant genetic structure revealed by microsatellite data suggests the existence of present-day barriers to gene flow between the Atlantic and Pacific populations most likely due to the influence of the Agulhas Current retroflection on prey movements.

Ancient origin of endemic Iberian earth-boring dung beetles (Geotrupidae).

The earth-boring dung beetles belong to the family Geotrupidae that includes more than 350 species classified into three subfamilies Geotrupinae, Lethrinae, and Taurocerastinae, mainly distributed across temperate regions. Phylogenetic relationships within the family are based exclusively on morphology and remain controversial. In the Iberian Peninsula there are 33 species, 20 of them endemic, which suggests that these lineages might have experienced a radiation event. The evolution of morphological adaptations to the Iberian semi-arid environments such as the loss of wings (apterism) or the ability to exploit alternative food resources is thought to have promoted diversification. Here, we present a phylogenetic analysis of 31 species of Geotrupidae, 17 endemic to the Iberian Peninsula, and the remaining from southeastern Europe, Morocco, and Austral South America based on partial mitochondrial and nuclear gene sequence data. The reconstructed maximum likelihood and Bayesian inference phylogenies recovered Geotrupinae and Lethrinae as sister groups to the exclusion of Taurocerastinae. Monophyly of the analyzed geotrupid genera was supported but phylogenetic relationships among genera were poorly resolved. Ancestral character-state reconstruction of wing loss evolution, dating, and diversification tests altogether showed neither evidence of a burst of cladogenesis of the Iberian Peninsula group nor an association between apterism and higher diversification rates. Loss of flight did not accelerate speciation rates but it was likely responsible for the high levels of endemism of Iberian geotrupids by preventing their expansion to central Europe. These Iberian flightless beetle lineages are probably paleoendemics that have survived since the Tertiary in this refuge area during Plio-Pleistocene climatic fluctuations by evolving adaptations to arid and semi-arid environments.

Evolution at a Different Pace: Distinctive Phylogenetic Patterns of Cone Snails from Two Ancient Oceanic Archipelagos

Ancient oceanic archipelagos of similar geological age are expected to accrue comparable numbers of endemic lineages with identical life history strategies, especially if the islands exhibit analogous habitats. We tested this hypothesis using marine snails of the genus Conus from the Atlantic archipelagos of Cape Verde and Canary Islands. Together with Azores and Madeira, these archipelagos comprise the Macaronesia biogeographic region and differ remarkably in the diversity of this group. More than 50 endemic Conus species have been described from Cape Verde, whereas prior to this study, only two nonendemic species, including a putative species complex, were thought to occur in the Canary Islands. We combined molecular phylogenetic data and geometric morphometrics with bathymetric and paleoclimatic reconstructions to understand the contrasting diversification patterns found in these regions. Our results suggest that species diversity is even lower than previously thought in the Canary Islands, with the putative species complex corresponding to a single species, Conus guanche. One explanation for the enormous disparity in Conus diversity is that the amount of available habitat may differ, or may have differed in the past due to eustatic (global) sea level changes. Historical bathymetric data, however, indicated that sea level fluctuations since the Miocene have had a similar impact on the available habitat area in both Cape Verde and Canary archipelagos and therefore do not explain this disparity. We suggest that recurrent gene flow between the Canary Islands and West Africa, habitat losses due to intense volcanic activity in combination with unsuccessful colonization of new Conus species from more diverse regions, were all determinant in shaping diversity patterns within the Canarian archipelago. Worldwide Conus species diversity follows the well-established pattern of latitudinal increase of species richness from the poles towards the tropics. However, the eastern Atlantic revealed a striking pattern with two main peaks of Conus species richness in the subtropical area and decreasing diversities toward the tropical western African coast. A Random Forests model using 12 oceanographic variables suggested that sea surface temperature is the main determinant of Conus diversity either at continental scales (eastern Atlantic coast) or in a broader context (worldwide). Other factors such as availability of suitable habitat and reduced salinity due to the influx of large rivers in the tropical area also play an important role in shaping Conus diversity patterns in the western coast of Africa.

Salamandra salamandra (Amphibia: Caudata: Salamandridae) in Portugal: not all black and yellow

The fire salamander complex is quite diverse in the Iberian Peninsula where nine subspecies of Salamandra salamandra are currently recognized. Here, we analysed the geographical distribution of the subspecies S. s. gallaica and S. s. crespoi using partial sequences of the mitochondrial cytochrome b gene of 168 individuals from 12 locations in Portugal. Our results support the existence of a deep lineage divergence between the two subspecies, with non-overlapping geographical distributions except in two contact zones: one in Sesimbra on the western coast, and another in Alcoutim on the southeastern border with Spain. Moreover, S. s. crespoi displays signs of gene flow among the sampled locations whereas S. s. gallaica shows evidence of some restriction to gene flow. Present-day genetic make-up of S. s. gallaica and S. s. crespoi is a result of past historical events, fine-tuned by contemporary Iberian geoclimate. Humid mountain areas were found to harbour increased genetic diversity possibly acting as past refugia during drier interglacial periods. To analyse wider geographical patterns and lineage splitting events within S. salamandra we performed a Bayesian dating analysis completing our data set with previously published sequences. The observed divergences were associated to successive biogeographic scenarios, and to other Iberian species showing similar trends.