Assisted gene flow using cryopreserved sperm in critically endangered coral

Abstract

Assisted gene flow (AGF) is a conservation intervention to accelerate species adaptation to climate change by importing genetic diversity into at-risk populations. Corals exemplify both the need for AGF and its technical challenges; corals have declined in abundance, suffered pervasive reproductive failures, and struggled to adapt to climate change, yet mature corals cannot be easily moved for breeding, and coral gametes lose viability within hours. Here, we report the successful demonstration of AGF in corals using cryopreserved sperm that was frozen for 2 to 10 y. We fertilized Acropora palmata eggs from the western Caribbean (Curaçao) with cryopreserved sperm from genetically distinct populations in the eastern and central Caribbean (Florida and Puerto Rico, respectively). We then confirmed interpopulation parentage in the Curaçao-Florida offspring using 19,696 single-nucleotide polymorphism markers. Thus, we provide evidence of reproductive compatibility of a Caribbean coral across a recognized barrier to gene flow. The 6-mo survival of AGF offspring was 42%, the highest ever achieved in this species, yielding the largest wildlife population ever raised from cryopreserved material. By breeding a critically endangered coral across its range without moving adults, we show that AGF using cryopreservation is a viable conservation tool to increase genetic diversity in threatened marine populations.

Keywords: Acropora palmata; assisted gene flow; coral reproduction; cryopreservation; endangered species.

Fig. 1.

Study species, experimental crosses, and fertilization rates. (A) Study species: elkhorn coral A. palmata. (B) Map of the Caribbean Sea and genetically distinct A. palmata populations. Circles indicate the locations of gamete collection and cryopreservation of sperm samples from Florida (FL, n = 2 sires), Puerto Rico (PR, n = 5 sires), and Curaçao (CUR, n = 6 sires). FT sperm was cryopreserved in the year listed and thawed immediately before use in in vitro fertilization experiments with freshly collected eggs from Curaçao (n = 5 dams). Freshly collected sperm from Curaçao (n = 4 or 5 sires, depending on spawning night) was used for comparison. (C) Summary of in vitro crosses conducted to test the feasibility of AGF in A. palmata. (D) Mean fertilization success for A. palmata eggs from five donor colonies (dams) when mixed with each of the four sperm pools or a no-sperm control (n = 1 to 9 containers per dam × sperm pool cross; replicate egg containers were preferentially assigned to the three FT crosses and the two interpopulation (AGF) crosses based on the lower fertilization rates expected from FT sperm and the higher conservation value of the AGF crosses). Sperm concentration and handling details are summarized in SI Appendix, Table S2. For all FT treatments, fertilization was attempted at two different sperm concentrations (n = 1 to 6 containers per concentration per cross); this bracketing approach during gamete handling was employed to maximize the overall chance of achieving fertilization using cryopreserved material (SI Appendix, Tables S2 and S3 for details).

Fig. 2.

Survival and growth of A. palmata juveniles reared from cryopreserved sperm. Photographs of (A–D) 1- and (E–H) 6-mo-old juvenile colonies of the coral A. palmata, reared from cryopreserved (FT) or fresh sperm crossed with freshly collected eggs from Curaçao (n = 5 dams). FT sperm was collected in Florida (FL, n = 2 sires), Puerto Rico (PR, n = 5 sires), and Curaçao (CUR, n = 6 sires). Fresh sperm from Curaçao was used for comparison (n = 4 or 5 sires, depending on spawning night) (Scale bars, 1 cm). (A and E) CUR×FL (FT sperm), (B and F) CUR×PR (FT sperm), (C and G) CUR×CUR (FT sperm), and (D and H) CUR×CUR (Fresh sperm). Due to their conservation value, the interpopulation (AGF) juveniles (A, B, E, and F) were given increased care and more space per juvenile beginning at the time of settlement. White areas around the juveniles are spaces where the coral inhibited encroaching coralline algae or coralline algae was removed to facilitate coral growth. No differences were apparent between the four cohorts in juvenile polyp morphology or colony growth pattern. (I) Survival of juveniles by cohort at 1 and 6 mo after settlement (n = 233 to 3,577 initial settlers per cohort at n = 2 separate rearing facilities; data were pooled across facilities to determine overall survival at each time point). The overall higher survival of the interpopulation (AGF) juveniles reflects the greater care directed to these cohorts due to their conservation value (SI Appendix, Table S4).

Fig. 3.

Parentage analysis of A. palmata juveniles reared from cryopreserved sperm demonstrating interpopulation parentage and successful AGF. (A and B) Genetic admixture plots for sires, dams, and offspring. The offspring genotyped for this analysis were drawn from two cohorts: CUR×FL (FT sperm, n = 15 offspring bred from n = 5 dams and n = 2 sires) and CUR×CUR (FT sperm, n = 15 offspring bred from n = 5 dams and n = 6 sires). The dataset also includes both of the known sires from Florida and five of the known and putative dams from Curaçao. The bars represent the probability of assignment (x-axis) for each individual (y-axis) to each of K = 2 genetic groups (A and B). (A) Probability of assignment using the full set of 19,696 genotyping SNPs for K = 2 populations. (B) Probability of assignment using only SNPs fixed between the Florida versus Curaçao parents (n = 41) for K = 2 populations. In (A and B), one of the juveniles from the CUR×FL cohort had a higher than expected probability of assignment to CUR (orange cluster) and is thought to be the result of self-fertilization or a labeling error (denoted with a *). (C and D) Triad assignment plots for the juveniles genotyped and analyzed Above. The data points represent the genetic dissimilarity among hypothetical triads of one sire, one dam, and one offspring. (C) Assignment plot of triads with FL sires, CUR dams, and only interpopulation (CUR×FL) offspring. A total of 13 of 15 juveniles from the interpopulation cross between FL sires (FT sperm) and CUR dams (fresh eggs) were successfully assigned to two specific parents in the sample set, shown by data points below the 0.0118 Gower genetic dissimilarity threshold. (D) Assignment plot of triads using FL sires, CUR dams, and only intrapopulation (CUR×CUR) offspring. As expected, no juveniles in the CUR×CUR cohort were assigned CUR×FL parentage (SI Appendix, Table S5 for details). (E) PCA of genetic structure among all coral dams, sires, and offspring genotyped in the analyses Above. PCA analysis was conducted using all 19,696 genotyping SNPs (R Package SNPrelate). All CUR×CUR offspring clustered with the CUR dams, while CUR×FL offspring formed a cluster in between their known FL sires and the known and putative CUR dams. As in (A and B), one CUR×FL juvenile clustered with the CUR×CUR offspring instead of with the other CUR×FL offspring, likely the result of selfing or mislabeling (denoted with a *).