Thermotolerance and regeneration in the brittle star species complex Ophioderma longicauda: A preliminary study comparing lineages and Mediterranean basins

Global warming is expected to change marine species distributions; it is thus critical to understand species current thermotolerance. The brittle star species complex Ophioderma longicauda comprises a broadcast spawning lineage L1 and a brooding lineage L3. We collected L1 specimens from Marseilles and Crete, and L3 specimens from Crete. We monitored survival, autotomy and arm regeneration at 17, 26 and 30 °C during 14 weeks. Globally O. longicauda showed good resistance to elevated temperatures compared to other published studies on ophiuroids. The L3 sample displayed a better thermotolerance than L1 samples. Yet, more research is needed to establish whether these differences are due to lineages, geographic origin, or random effects. We provided for the first time individual regeneration trajectories, and showed that regeneration followed a growth curve and was highly influenced by temperature in both lineages. Our results highlight the importance of taking into account the presence of cryptic species when studying the potential effects of global warming.     

Reproductive ecology of Caribbean reef corals

The last decade has seen a resurgence of interest in the processes of sexual reproduction by scleractinian reef corals. Earlier investigations had focused fortuitously on brooding (planulating) species, which resulted in the general misconception that brooding was the main form of larval development of reef corals. More recent work on Indo-Pacific species has shown broadcast spawning and short annual reproductive periods to predominate. This report presents the reproductive patterns of eleven Caribbean coral species and attempts to explain the adaptive features and selective pressures that have led to the evolution of the four reproductive patterns described to date: (a) hermaphroditic broadcasters; (b) gonochoric broadcasters; (c) hermaphroditic broadcasters; (b) gonochoric brooders. Both (a) and (b) correlate with large colony size and short annual spawning periods; and (c) and (d) correlate with small colony size, multiple planulating cycles per year, and occupation of unstable habitats. Selection for outcrossing between long-lived individuals is proposed as the reason for gonochorism and for synchronous spawning of hermaphroditic broadcasters, and also for the large amount of sperm produced by hermaphroditic brooders. Selection for high rates of local recruitment is proposed as the force behind the evolution of brooding by species inhabiting unstable habitats and suffering high rates of adult mortality.     

Is the Scotia Sea a centre of Antarctic marine diversification? Some evidence of cryptic speciation in the circum-Antarctic bivalve <Emphasis Type="Italic">Lissarca notorcadensis</Emphasis> (Arcoidea: Philobryidae)

The bivalve Lissarca notorcadensis is one of the most abundant species in Antarctic waters and has colonised the entire Antarctic shelf and Scotia Sea Islands. Its brooding reproduction, low dispersal capabilities and epizoic lifestyle predict limited gene flow between geographically isolated populations. Relationships between specimens from seven regions in the Southern Ocean and outgroups were assessed with nuclear 28S rDNA and mitochondrial cytochrome oxidase subunit I (COI) genes. The 28S dataset indicate that while Lissarca appears to be a monophyletic genus, there is polyphyly between the Limopsidae and Philobryidae. Thirteen CO1 haplotypes were found, mostly unique to the sample regions, and two distinct lineages were distinguished. Specimens from the Weddell and Ross Sea form one lineage while individuals from the banks and islands of the Scotia Sea form the other. Within each lineage, further vicariance was observed forming six regionally isolated groups. Our results provide initial evidence for reproductively isolated populations of L. notorcadensis. The islands of the Scotia Sea appear to act as centres of speciation in the Southern Ocean.     

Difference in larval type explains patterns of nonsynonymous substitutions in two ancient paralogs of the histone H3 gene in sea stars

SUMMARY Paralogous genes frequently show differences in patterns and rates of substitution that are typically attributed to different selection regimes, mutation rates, or local recombination rates. Here, two anciently diverged paralogous copies of the histone H3 gene in sea stars, the tandem‐repetitive early‐stage gene and a newly isolated gene with lower copy number that was termed the “putative late‐stage histone H3 gene” were analyzed in 69 species with varying mode of larval development. The two genes showed differences in relative copy number, overall substitution rates, nucleotide composition, and codon usage, but similar patterns of relative nonsynonymous substitution rates, when analyzed by the d_N/d_S ratio. Sea stars with a nonpelagic and nonfeeding larval type (i.e., brooding lineages) were observed to have d_N/d_S ratios that were larger than for nonbrooders but equal between the two paralogs. This finding suggested that demographic differences between brooding and nonbrooding lineages were responsible for the elevated d_N/d_S ratios observed for brooders and refuted a suggestion from a previous analysis of the early‐stage gene that the excess nonsynonymous substitutions were due to either (1) gene expression differences at the larval stage between brooders and nonbrooders or (2) the highly repetitive structure of the early‐stage histone H3 gene.     

Evidence of horizontal gene transfer between land plant plastids has surprising conservation implications

Background and AimsHorizontal gene transfer (HGT) is an important evolutionary mechanism because it transfers genetic material that may code for traits or functions between species or genomes. It is frequent in mitochondrial and nuclear genomes but has not been demonstrated between plastid genomes of different green land plant species.MethodsWe Sanger-sequenced the nuclear internal transcribed spacers (ITS1 and 2) and the plastid rpl16 G2 intron (rpl16). In five individuals with foreign rpl16 we also sequenced atpB-rbcL and trnL_UAA-trnF_GAA.Key ResultsWe discovered 14 individuals of a moss species with typical nuclear ITSs but foreign plastid rpl16 from a species of a distant lineage. None of the individuals with three plastid markers sequenced contained all foreign markers, demonstrating the transfer of plastid fragments rather than the entire plastid genome, i.e. entire plastids were not transferred. The two lineages diverged 165–185 Myr BP. The extended time interval since lineage divergence suggests that the foreign rpl16 is more likely explained by HGT than by hybridization or incomplete lineage sorting.ConclusionsWe provide the first conclusive evidence of interspecific plastid-to-plastid HGT among land plants. Two aspects are critical: it occurred at several localities during the massive colonization of recently disturbed open habitats that were created by large-scale liming as a freshwater biodiversity conservation measure; and it involved mosses whose unique life cycle includes spores that first develop a filamentous protonema phase. We hypothesize that gene transfer is facilitated when protonema filaments of different species intermix intimately when colonizing disturbed early succession habitats.     

Phylogeography of the Coastal Mosquito Aedes togoi across Climatic Zones: Testing an Anthropogenic Dispersal Hypothesis

The coastal mosquito Aedes togoi occurs more or less continuously from subarctic to subtropic zones along the coasts of the Japanese islands and the East Asian mainland. It occurs also in tropical Southeast Asia and the North American Pacific coast, and the populations there are thought to have been introduced from Japan by ship. To test this hypothesis, the genetic divergence among geographic populations of A. togoi was studied using one mitochondrial and three nuclear gene sequences. We detected 71 mitochondrial haplotypes forming four lineages, with high nucleotide diversity around temperate Japan and declining towards peripheral ranges. The major lineage (L1) comprised 57 haplotypes from temperate and subarctic zones in Japan and Southeast Asia including southern China and Taiwan. Two other lineages were found from subtropical islands (L3) and a subarctic area (L4) of Japan. The Canadian population showed one unique haplotype (L2) diverged from the other lineages. In the combined nuclear gene tree, individuals with mitochondrial L4 haplotypes diverged from those with the other mitochondrial haplotypes L1—L3; although individuals with L1—L3 haplotypes showed shallow divergences in the nuclear gene sequences, individuals from Southeast Asia and Canada each formed a monophyletic group. Overall, the genetic composition of the Southeast Asian populations was closely related to that of temperate Japanese populations, suggesting recent gene flow between these regions. The Canadian population might have originated from anthropogenic introduction from somewhere in Asia, but the possibility that it could have spread across the Beringian land bridge cannot be ruled out.     

Recent relaxation of purifying selection on the tandem-repetitive early-stage histone H3 gene in brooding sea stars

Patterns of nucleotide substitution differ between marine species that have a pelagic feeding (planktotrophic) larval stage and related species that lack such a stage, for both adaptive and non-adaptive reasons. Here, patterns of nucleotide and inferred amino acid substitution are analyzed for the tandem-repetitive early-stage histone H3 gene in 36 sea star species of the order Forcipulatida with documented larval habitat. The relative rate of nonsynonymous substitution (expressed as ω = d_N/d_S) was significantly higher in lineages with a brooded non-feeding (lecithotrophic) larval form than in lineages with a planktotrophic larval form. There was also a significant excess of conservative over radical substitutions. The increase in ω for brooders as compared to non-brooders was much greater than for previously analyzed mitochondrial sequences in echinoderms. These data are consistent with the hypothesis that purifying selection on this gene has been relaxed in brooding lineages compared to non-brooding lineages. The hypotheses of adaptive or neutral evolution are less plausible, although recent pseudogenization following a period of relaxed purifying selection could also explain the results.     

Genetic differentiation of <Emphasis Type="Italic">Ganaspis brasiliensis</Emphasis> (Hymenoptera: Figitidae) from East and Southeast Asia

Ganaspis brasiliensis (Ihering) (Hymenoptera: Figitidae: Eucoilinae) is a Drosophila parasitoid that has often been misidentified as G. xanthopoda (Ashmead) in recent studies. This study aims to clarify genetic differentiation of G. brasiliensis based on the nucleotide sequences of the mitochondrial cytochrome oxidase subunit 1 (CO1) gene and three nuclear DNA regions, the inter-transcribed spacers 1 and 2 (ITS1 and ITS2) and putative 60S ribosomal protein L37 (RpL37), as well as crossing experiments. Four lineages are recognized in individuals assigned as G. basiliensis by morphology, (1) individuals occurring in Japan and probably South Korea, (2) individuals from a small subtropical island of Japan, Iriomote-jima, (3) individuals from temperate lowlands of Japan and high altitude areas of Southeast Asia, and (4) individuals occurring widely in Asia, America, Hawaii and Africa. The first lineage is a specialist of Drosophila suzukii (Matsumura), a pest of fresh fruit, and also the fourth lineage has a capacity to parasitize this pest species. The first, third and fourth lineages occur sympatrically at least in Tokyo. The third and fourth lineages differed in mate choice and host use to some extent, but post-mating isolation between them was almost absent.     

Phylogeography of the Lacerta viridis complex: mitochondrial and nuclear markers provide taxonomic insights

Based on broad, nearly rangewide sampling, we reanalysed the phylogeography of the Lacerta viridis complex using the mitochondrial cytochrome b gene and the intron 7 of the nuclear β‐fibrinogen gene. Using the mitochondrial marker, we identified in phylogenetic analyses 10 terminal clades clustering in four deeply divergent main lineages whose relationships are weakly resolved. These lineages correspond to Lacerta bilineata, L. viridis, the previously identified Adriatic or West Balkan lineage and a newly discovered fourth lineage from the Anatolian Black Sea coast and the south‐eastern Balkan Peninsula. Except for the latter lineage, there is considerable phylogeographic structuring in each lineage, with higher diversity in the south of the distribution ranges. This pattern indicates the existence of two distinct microrefugia in the Italian Peninsula and Sicily and of up to seven microrefugia in the Balkan Peninsula, but of only one refugium along the Black Sea coast of Anatolia. We identified secondary contact zones of the main lineages and of terminal clades within these lineages. However, most of the formerly described putative contact zone of L. bilineata and L. viridis turned out to be a contact zone between the Adriatic lineage and L. viridis, but L. bilineata seems to be involved only marginally. Our nuclear marker could not unambiguously resolve whether there is gene flow in contact zones. Thus, further research is necessary to decide whether the four main lineages are conspecific or whether they represent distinct biological species. We restrict the name L. v. meridionalis to the newly identified genetic lineage from Turkey and south‐eastern Europe, synonymize some previously recognized taxa and suggest a tentative nomenclature for the L. viridis complex.     

The hidden diversity of Coleodactylus amazonicus (Sphaerodactylinae,Gekkota) revealed by molecular data

Coleodactylus amazonicus, a small leaf-litter diurnal gecko widely distributed in Amazon Basin, has been considered a single species with no significant morphological differences between populations along its range. A recent molecular study, however, detected large genetic differences between populations of central Amazonia and those in the easternmost part of the Amazon Basin, suggesting the presence of taxonomically unrecognised diversity. In this study, DNA sequences of three mitochondrial (16S, cytb, and ND4) and two nuclear genes (RAG-1, c-mos) were used to investigate whether the species currently identified as C.amazonicus contains morphologically cryptic species lineages. The present phylogenetic analysis reveals further genetic subdivision including at least five potential species lineages, restricted to northeastern (lineage A), southeastern (lineage B), central-northern (lineage E) and central-southern (lineages C and D) parts of Amazon Basin. All clades are characterized by exclusive groups of alleles for both nuclear genes and highly divergent mitochondrial haplotype clades, with corrected pairwise net sequence divergence between sister lineages ranging from 9.1% to 20.7% for the entire mtDNA dataset. Results of this study suggest that the real diversity of “C. amazonicus” has been underestimated due to its apparent cryptic diversification.     

Lineage-specific group II intron gains and losses of the mitochondrial rps3 gene in gymnosperms

According to PCR assays and sequencing, we now report the shared presence of two rps3 introns, namely the rps3i74 and the rps3i249, in the mitochondria of all the classes representing the surviving lineages of gymnosperms, and unveil several lineages experiencing intron loss.Interestingly, the rps3 intron gains and losses within the four groups of gymnosperms let us sort out the Pinaceae and the non-Pinaceae into intron (+)- and intron (?)-lineages, respectively. Worthy of mention is also the finding that only Gnetum within the Gnetales harbours both the rps3 introns.This intron distribution pattern is consistent with the hypothesis that the two rps3 introns were likely present in the common ancestor of the seed plants and, then, independently lost in the non-Pinaceae during gymnosperm evolution.The derived secondary structural model of the novel group IIA intron improves our understanding of the significance and origin of the extraordinary length polymorphisms observed among rps3i249 orthologs.Despite the remarkable structural plasticity to adopt and reject introns, the rps3 mRNAs undergo accurate processing by splicing and extensive editing in gymnosperm mitochondria.This study provides additional insights into the evolutionarily high dynamics of mitochondrial introns which may come and go in closely related plant species.The turnover of the mitochondrial rps3 group II introns seen among lineages of seed plants further suggests that these introns might be an additional signature to discriminate between particularly cryptical taxonomic groups for which there is a need of a further evaluation of their evolutionary affiliation.     

A three‐way contact zone between forms of Patella rustica (Mollusca: Patellidae) in the central Mediterranean Sea

Previous studies have reported the occurrence of three differentiated mtDNA lineages within Patella rustica in the Mediterranean Sea. Two hypotheses have been proposed to explain these observations: (1) the maintenance of ancestral polymorphism within a single species; (2) the occurrence of cryptic species not identified previously. To distinguish between these hypotheses, we screened the genetic variability at nine allozyme loci, an intron from the α‐amylase gene and a mitochondrial gene for 187 individuals of P. rustica sampled from seven Mediterranean localities. Eight additional localities were screened for the last two markers to place the differentiated lineages in a clear geographic context. Our results demonstrate that the three mtDNA lineages correspond to three distinct nuclear genotype clusters and provide further details on their distribution: the cluster corresponding to the mtDNA lineage from the Atlantic and western Mediterranean extends as far as the south coast of Italy, whereas the remaining two clusters occur in sympatry in the eastern Mediterranean. One of the eastern Mediterranean clusters is highly differentiated and seems to be reproductively isolated from the codistributed form; we therefore suggest that it corresponds to a new species. The remaining two clusters are less differentiated and form a contact zone across south Italian shores. This three‐way contact zone constitutes an interesting model for the study of speciation in the marine realm. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 154–169.     

Is reproductive strategy a key factor in understanding the evolutionary history of Southern Ocean Asteroidea (Echinodermata)?

Life traits such as reproductive strategy can be determining factors of species evolutionary history and explain the resulting diversity patterns. This can be investigated using phylogeographic analyses of genetic units. In this work, the genetic structure of five asteroid genera with contrasting reproductive strategies (brooding: Diplasterias, Notasterias and Lysasterias versus broadcasting: Psilaster and Bathybiaster) was investigated in the Southern Ocean. Over 1,400 mtDNA cytochrome C oxidase subunit I (COI) sequences were analysed using five species delineation methods (ABGD, ASAP, mPTP, sGMYC and mGMYC), two phylogenetic reconstructions (ML and BA), and molecular clock calibrations, in order to examine the weight of reproductive strategy in the observed differences among phylogeographic patterns. We hypothesised that brooding species would show higher levels of genetic diversity and species richness along with a clearer geographic structuring than broadcasting species. In contrast, genetic diversity and species richness were not found to be significantly different between brooders and broadcasters, but broadcasters are less spatially structured than brooders supporting our initial hypothesis and suggesting more complex evolutionary histories associated to this reproductive strategy. Broadcasters' phylogeography can be explained by different scenarios including deep‐sea colonisation routes, bipolarity or cosmopolitanism, and sub‐Antarctic emergence for the genus Bathybiaster; Antarctic‐ New Zealand faunal exchanges across the Polar Front for the genus Psilaster. Brooders' phylogeography could support the previously formulated hypothesis of a past trans‐Antarctic seaway established between the Ross and the Weddell seas during the Plio‐Pleistocene. Our results also show, for the first time, that the Weddell Sea is populated by a mixed asteroid fauna originating from both the East and West Antarctic.     

Nuclear DNA recapitulates the cryptic mitochondrial lineages of Lumbricus rubellus and suggests the existence of cryptic species in an ecotoxological soil sentinel

Mitochondrial DNA analysis has revealed two distinct phylogenetic lineages within the ecotoxological sentinel earthworm model Lumbricus rubellus Hoffmeister, 1843. The existence of these lineages could complicate ecotoxicological studies that use the species as a sentinel for soil contamination testing, as they may respond differently to contamination; however, as mitochondrial haplotypes are not always expected to segregate in the same way as chromosomal DNA in natural populations, we further investigated this issue by using nuclear DNA markers (microsatellites) to measure genetic diversity, differentiation, and gene flow in sympatric populations of the two L. rubellus lineages at two sites in South Wales. Our results show that sympatric populations of the two lineages are more genetically differentiated than geographically distant populations of the same lineage, and Bayesian clustering analysis revealed no evidence of gene flow between the lineages at either site. Additionally, DNA sequencing of these microsatellite loci uncovered substantial differentiation between lineages at homologous flanking regions. Overall our findings indicate a high degree of nuclear genetic differentiation between the two lineages of L. rubellus, implying reproductive isolation at the two study sites and therefore the potential existence of cryptic species. The existence of two cryptic taxa has major implications for the application of L. rubellus as an ecotoxicological sentinel. It may therefore be necessary to consider the lineages as separate taxa during future ecotoxicological studies. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 780–795.     

Multilocus phylogeography of the Patagonian lizard complex Liolaemus kriegi (Iguania: Liolaemini)

This study presents a detailed phylogeographical analysis of one of the most conspicuous groups of lizards in northwestern Patagonia, the Liolaemus kriegi complex. This region is geographically very complex as a result of Andean orogeny and subsequent volcanism coupled with a long history of glaciations and climatic changes. For 247 individuals we sequenced one mitochondrial gene (cytochrome b) and for a subset we sequenced another mitochondrial gene [12S ribosomal RNA (12S)] and two nuclear fragments [kinesin family member 24 (KIF24) and BA3 ribosomal RNA (BA3)]. We obtained gene trees and mitochondrial and nuclear haploytpe networks, and estimated genetic distances between the main lineages and basic molecular diversity indices. We also performed spatial analysis of molecular variance (SAMOVA) and Bayesian Skyline Plot (BSP) analyses, and concordant patterns from different lines of evidence permitted delimitation of seven lineages: two described species, Liolaemus buergeri and Liolaemus tregenzai; four candidate species, Liolaemus sp. A, Liolaemus sp. B, Liolaemus sp. C, and Liolaemus sp. D; and one lineage that includes all individuals from the geographical range of Liolaemus ceii and L. kriegi, referred to as L. kriegi + L. ceii. We discuss the evolutionary processes that may contribute to the origin of these lineages and their taxonomic and conservation implications. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 256–269.     

Integrative taxonomy of Pseudolepeophtheirus longicauda (Crustacea: Copepoda: Caligidae) parasitic on Platichthys stellatus (Actinopterygii: Pleuronectidae)

Based on specimens from the gill cavities of one Platichthys stellatus individual collected in the Sea of Japan, we investigated the taxonomic status of the enigmatic caligid genus Pseudolepeophtheirus and its type species, Pseudolepeophtheirus longicauda. In a maximum likelihood (ML) tree based on 18S rRNA gene sequences, the sequence from our sample was nested in a well-supported Lepeophtheirus clade, along with the type species, confirming that Pseudolepeophtheirus should be considered a junior synonym of Lepeophtheirus; our morphological data support this synonymy. Although a previous study had synonymized Pseudolepeophtheirus longicauda with Lepeophtheirus parvicruris, we found that the former differs morphologically from the latter in having a short leg-4 exopod, with the articulation between the first and second segments not evident (the shape of the posterior striated membrane on the leg-2 intercoxal sclerite also differs between two species), and detected slight differences in 18S rRNA sequences between two taxa. We thus concluded that this synonymy is invalid, and reinstate Lepeophtheirus longicauda as a valid species. A ML analysis of COI sequences from Pl. stellatus (the host fish for both L. longicauda and L. parvicruris) showed the host species to comprise distinct northwestern- and northeastern-Pacific clades. Lepeophtheirus longicauda is distributed in the northwestern Pacific and L. parvicruris in the northeastern Pacific, indicating co-divergence of the two copepod species with the host lineages.     

Species boundaries in the Himantura uarnak species complex (Myliobatiformes: Dasyatidae)

Samples of the ‘Himantura uarnak’ species complex (H. leoparda, H. uarnak, H. undulata under their current definitions), mostly from the Coral Triangle, were analyzed using nuclear markers and mitochondrial DNA sequences. Genotypes at five intron loci showed four reproductively isolated clusters of individuals. The COI sequences showed four major mitochondrial lineages, each diagnostic of a cluster as defined by nuclear markers. No mitochondrial introgression was detected. The average Kimura-2 parameter nucleotide distance separating clades was 0.061–0.120 (net: 0.055–0.114), while the distance separating individuals within a clade was 0.002–0.008. Additional, partial cytochrome-b gene sequences were used to link these samples with previously published sequences of reference specimens of the three nominal species. One of the clusters was identified as H. undulata and another one, as H. uarnak, while two cryptic species were uncovered within the recently-described H. leoparda, challenging the current morphology-based taxonomy of species within the H. uarnak species complex.     

Molecular data and species distribution models reveal the Pleistocene history of the mayfly Ameletus inopinatus (Ephemeroptera: Siphlonuridae)1

1. We investigated the Pleistocene and Holocene history of the rare mayfly Ameletus inopinatus EATON 1887 (Ephemeroptera: Siphlonuridae) in Europe. We used A. inopinatus as a model species to explore the phylogeography of montane, cold‐tolerant aquatic insects with arctic–alpine distributions. 2. Using species distribution models, we developed hypotheses about the species demographic history in Central Europe and the recolonisation history of Fennoscandia. We tested these hypotheses using mitochondrial cytochrome oxidase I (mtCOI) sequence data and compared our genetic results with previously generated microsatellite data to explore genetic diversity distributions of A. inopinatus. 3. We observed old lineages, deep splits and almost complete lineage sorting of mtCOI sequences among mountain ranges. These results support a periglacial survival, i.e. persistence at the periphery of Pleistocene glaciers in Central Europe. 4. There was strong differentiation between the Fennoscandian and all other populations, indicating that Fennoscandia was recolonised from a refugium not accounted for in our sampling. High degrees of population genetic structure within the northern samples suggest that Fennoscandia was recolonised by more than one lineage. However, this structure was not apparent in previously published microsatellite data, consistent with secondary contact without sexual incompatibility or with sex‐biased dispersal. 5. Our demographic analyses indicate that (i) the separation of northern and Central European lineages occurred during the early Pleistocene; (ii) Central European populations have persisted independently throughout the Pleistocene and (iii) the species extended its range about 150 000 years ago.     

Phylogenetic analysis of the Sphaeriidae (Mollusca: Bivalvia) based on partial mitochondrial 16s rDNA gene sequences

Abstract. We have constructed molecular phylogenetic trees for members of the Sphaeriidae in order to test proposed generic level relationships, and to reconstruct the evolutionary pattern of parental care, in this exclusively freshwater family of heterodont bivalves. An ∼480 nucleotide fragment of the mitochondrial large ribosomal subunit (16s rDNA) was sequenced for 4 corbiculid outgroups in addition to 19 sphaeriid ingroup taxa. Ingroup species were obtained from North and South America, Europe, and Australasia and included representatives of the main sphaeriid genera. Our analyses support four primary conclusions: 1) the Sphaeriinae are robustly monophyletic with respect to Eupera platensis ; 2) the genus Pisidium is paraphyletic and P. sterkianum is sister to the 17 other sphaeriine taxa in our dataset; 3) synchronous brooding is the ancestral reproductive pattern in the Sphaeriinae; 4) the sequential brooders form a clade in which Musculium taxa are monophyletic and nested among lineages of Sphueriunz. Our gene trees reveal an evolutionary progression in parental care complexity from the relatively simple pattern in the Euperinae, to the origin of brood sacs and of extraoogonial embryonic nutrition in the common ancestor of the Sphaeriinae, and ultimately to the development of sequential brooding in Sphaerium/Musculium taxa.