A synthetic phylogeny of freshwater crayfish: insights for conservation

Freshwater ecosystems cover only 0.8% of Earth’s surface but harbor nearly 6% of described species and face multiple threats, including overexploitation, pollution, flow modification, habitat degradation, and invasive species. Regions like the southeastern United States exhibit high freshwater biodiversity and endemism, but the fragmented nature of habitats both promotes speciation and increases susceptibility to habitat loss and limited dispersal, contributing to accelerated extinction rates comparable to tropical rainforests. Crayfish are key ecological players and cultural/economic resources within these endangered systems and have been considered keystone species through consumer activity, predation, sediment bioturbation, and enhanced organic matter processing. They also act as ecological engineers in some terrestrial systems. Over 30% of known crayfish species are endangered or at risk. There are over 600 extant species across three families (Parastacidae, Cambaridae, Astacidae) and 30 genera, distributed worldwide in temperate regions except Antarctica and continental Africa (with an endemic genus in Madagascar). Centers of diversity include southeast Australia/Tasmania (Parastacidae) and the southeastern United States (Cambaridae). Crayfish are suitable for phylogenetic synthesis due to robust taxonomy and extensive phylogenetic work and have been recently assessed by the IUCN Red List. This study synthesizes recent crayfish phylogenies with taxonomy to estimate a comprehensive synthesis tree, maps IUCN status to test associations with clades and taxonomic groups, constructs a branch-length phylogram and a fossil-calibrated chronogram to estimate diversification and extinction rates, and integrates phylogenetic diversity (PD) with threat status to identify evolutionarily distinct and globally endangered species via EDGE analyses. The study demonstrates how combining taxonomy with synthetic phylogeny can support conservation prioritization based on PD and endangerment.

Phylogenetic analyses and synthesis: Synthetic tree estimation involved uploading 20 published, rooted phylogenies (Newick format) to The Open Tree of Life Study Curator, mapping taxa to a user-curated Arthropoda taxonomy (OTT2.2) compiled from WoRMS, GenBank, and GBIF, and exporting curated NeXML files. Source trees and the taxonomy were merged in treemachine into a tree alignment graph according to a predefined study order to generate a synthetic tree. Taxa lacking source-tree representation are included via the taxonomy backbone.

Crayfish phylogeny with branch lengths: To obtain branch lengths for PD analyses, the authors built a supermatrix phylogram with PHLAWD from GenBank for species-level terminals (excluding intraspecific variants and checking COI for intact ORFs and avoiding nuclear mitochondrial pseudogenes). Loci included mitochondrial 12S, 16S, COI and nuclear 18S, 28S. Alignments were performed with MAFFT v7.130b; poorly aligned regions were removed with GBLOCKS under least stringent settings. Two lobster outgroups (Homarus americanus, Enoplometopus occidentalis) were used. Partitioning and models were selected with PartitionFinder v1.1.1 using BIC across seven subsets (12S, 16S, 18S, 28S, COI 1st, 2nd, 3rd codon positions). A maximum-likelihood tree was inferred in GARLI 2.0 with multiple random-start searches; node support assessed via 100 non-parametric bootstrap replicates.

Chronogram estimation: Divergence times were inferred with penalized likelihood in r8s using the ML tree, with six fossil calibrations spanning Mid Triassic (~225 Ma), Late Jurassic (~145 Ma), Early Cretaceous (~135 Ma), and Eocene (~40 Ma), following prior crayfish studies. The smoothing parameter was chosen via cross-validation.

Diversification rates through time: To test constancy of diversification, the Monte Carlo Constant Rates (MCCR) test was applied to branching times, accounting for non-random missing taxa by simulating constant-rate birth-death chronograms in CORSIM with 40% missing taxa and a youngest genus age of 9.95 Ma. The observed gamma statistic was compared to a null distribution of 1000 simulated trees in R (APE). Time-dependent diversification (birth-death) rate shifts were estimated by ML using TREEPAR, evaluating models with up to four shifts, computing rates at 1 Myr intervals while accounting for missing taxa, and comparing nested models via chi-squared tests (3 d.f. per added shift).

Conservation status, PD, EDGE/HEDGE: IUCN Red List v2013.2 categories were assigned per species. EDGE scores were computed from evolutionary distinctiveness (branch lengths weighted by descendant counts) combined with extinction probabilities assigned per IUCN class (LC 0.025, NT 0.05, VU 0.1, EN 0.2, CR 0.4). HEDGE scores were computed as expected contribution to future PD (probabilistic PD allowing extinction probabilities down to 0). Calculations were performed in MESQUITE with the Tuatara module. To assess regional PD patterns, species were coded for presence/absence across eight FADA freshwater ecozones based on IUCN range data. Phylogenetic species variability (PSV; independent of species richness) and its variance, and Faith’s PD, were computed in the R package picante from the chronogram (outgroups removed).

Phylogenetic synthesis: The curated synthesis produced 719 terminal taxa, including all 590 described freshwater crayfish species plus multiple population-level GenBank lineages; 387 taxa (about 60% of described species) have sequence data. Procambarus is the largest genus but poorly sampled genetically (31/178 species with data).

Molecular phylogeny: The supermatrix after GBLOCKS trimming comprised 5259 bp. The ML tree strongly supports reciprocal monophyly of Northern vs Southern Hemisphere clades (bootstrap 100). Parastacidae is strongly supported as monophyletic (BS 98), whereas Cambaridae and Astacidae are paraphyletic. Many large Northern Hemisphere genera (Orconectes, Procambarus, Cambarus) are not monophyletic; Southern Hemisphere genera are mostly monophyletic but with limited support among genera.

Diversification dynamics: The constant-rate Yule model was rejected. A three-rate birth-death model fit best with diversification-rate shifts at approximately 20 Ma and 4 Ma. Estimated parameters (per figure) indicate increasing extinction (death) toward the present, with birth decreasing then increasing at the present; turnover (μ/λ) is highest in the most recent interval. Reported interval-specific ML estimates include λ ≈ 6.08×10^-2, μ ≈ 3.27×10^-5 (μ/λ ≈ 5.38×10^-5); λ ≈ 8.28×10^-1, μ ≈ 2.63×10^-2 (μ/λ ≈ 3.18×10^-2); λ ≈ 5.64×10^-2, μ ≈ 1.53×10^-2 (μ/λ ≈ 2.71×10^-1).

Regional PD and PSV: Across FADA ecozones, the Nearctic exhibits the highest PD (11026.91), while the Neotropical has the highest PSV (0.74). Australasian PSV is high (0.72) due to long branches indicating few close relatives and substantial evolutionary history. The Afrotropical region (Malagasy genus with seven species) is least diverse (PSV 0.16; PD 266.54) and shows the greatest PSV variance, reflecting non-clocklike branch length heterogeneity among its few taxa. Additional PD/PSV values include Palearctic PD 861.7 with PSV 0.63.

EDGE/HEDGE prioritization: EDGE scores ranged from 35.54 to 0.36; HEDGE from 34.91 to 0.12. Fallicambarus hortoni had the highest EDGE and HEDGE scores. The top-ranking species are predominantly Australian lineages (Engaeus, Engaewa, Euastacus) and several North American cave Cambarus species (e.g., C. tartarus, C. laconensis, C. aculabrum).

Integrating phylogeny with IUCN threat status enables prioritization that preserves maximal phylogenetic diversity. The crayfish synthesis reveals significant taxonomy-phylogeny discordance, especially in North American taxa, where rapid recent radiations (short internodes) and limited informative morphology contribute to non-monophyly of several genera and paraphyly of families Cambaridae and Astacidae. Southern Hemisphere taxa generally show monophyletic genera and long terminal branches, possibly reflecting reduced recent diversification associated with Miocene climatic shifts (e.g., Antarctic Circumpolar Current formation) and subsequent aridification and glaciation.

From a conservation standpoint, the current phylogeny’s incomplete sampling (about 60%) and unresolved relationships limit confidence in absolute EDGE/HEDGE rankings, as missing species can shorten edge lengths and alter distinctiveness metrics. Nonetheless, results highlight regions and lineages harboring large amounts of unique evolutionary history and elevated threat, especially Australian parastacids and North American cave endemics.

Methodologically, reliance on a traditional five-locus marker set limits resolution within and among genera. Genomic-scale data (anchored hybrid enrichment, ultraconserved elements, transcriptomes) should improve backbone resolution and support within clades. Diversification analyses indicate non-constant rates with shifts around ~20 Ma and ~4 Ma, broadly coinciding with major paleoenvironmental events (onset of Antarctic Circumpolar Current and Northern Hemisphere glaciations), though precise timing and rates would benefit from Bayesian divergence dating with improved convergence on large trees. Future work combining phylogeny, ecology, morphology, and geography can model trait- or environment-dependent diversification and extinction risks to refine conservation actions.

This study synthesizes taxonomy and multiple phylogenetic sources to produce a comprehensive crayfish synthesis tree, a branch-length phylogram, and a fossil-calibrated chronogram, enabling assessments of diversification dynamics and conservation prioritization via PD, EDGE, and HEDGE metrics. Key insights include strong Southern vs Northern Hemisphere clades, monophyly of Parastacidae with paraphyly of Cambaridae and Astacidae, temporal shifts in diversification rates, and identification of evolutionarily distinct, threatened lineages concentrated in Australia and North American cave systems. Advancing crayfish conservation will require a reappraisal of taxonomy, broader species sampling, and adoption of genomic-scale phylogenetics to resolve relationships and accurately quantify evolutionary distinctiveness. Future research should produce a near-complete, well-resolved, time-calibrated phylogeny, integrate Bayesian dating approaches, and link trait and environmental data to diversification/extinction models to guide targeted conservation.

• Incomplete taxon sampling: approximately 40% of described crayfish species lack sequence data in the phylogeny, which can bias EDGE/HEDGE by inflating terminal edge lengths and affect PD/PSV estimates.
• Taxonomy-phylogeny discordance: Several genera (notably in Northern Hemisphere Cambaridae) are non-monophyletic, complicating placement of missing taxa and interpretation of clade-based conservation priorities.
• Limited marker set and resolution: Traditional five-locus datasets insufficiently resolve many inter- and intrageneric relationships; genomic data are needed.
• Synthetic tree lacks branch lengths: Taxonomy-only placements in the synthesis cannot be used directly for EDGE/HEDGE that require branch lengths.
• Divergence dating uncertainty: Penalized-likelihood point estimates lack full uncertainty quantification; Bayesian approaches on large trees faced convergence challenges.
• MCCR sensitivity: Constant-rate tests and diversification shift inferences can be sensitive to non-random sampling and assumptions about missing taxa, despite attempts to account for them.