Macroalgae cover up to 80% of the benthos along the western Antarctic Peninsula (WAP; Wiencke & Amsler Reference Wiencke, Amsler, Wiencke and Bischof2012). One of the most common and widespread members of the understory community is the red macroalga Plocamium sp. (Heiser et al. Reference Heiser, Amsler, McClintock, Shilling and Baker2020). It supports among the highest amphipod and gastropod densities and is protected from predation through highly diverse chemical defences (Heiser et al. Reference Heiser, Amsler, McClintock, Shilling and Baker2020). Haplotypic diversity, based on the mitochondrial cox1 barcode, showed some evidence of geographical structure as well as correlation with specific chemical defences (Shilling et al. Reference Shilling, Heiser, Amsler, McClintock and Baker2021). These coarse patterns of genetic diversity are insufficient to understand the processes structuring populations of Plocamium sp. along the WAP, necessitating the use of more polymorphic, nuclear loci, such as microsatellites.

Microsatellites have enabled the empirical quantification of the relative rates of selfing (i.e. self-fertilization) vs outcrossing (e.g. Winn et al. Reference Winn, Elle, Kalisz, Cheptou, Eckert and Goodwillie2011) and sexual vs asexual reproduction (e.g. Vallejo-Marín et al. Reference Vallejo-Marín, Dorken and Barrett2010), but studies have been restricted largely to angiosperms or animals, with far fewer investigations in macroalgae (Krueger-Hadfield et al. Reference Krueger-Hadfield, Guillemin, Destombe, Valero and Stoeckel2021). Plocamium sp., like many macroalgae, has a haploid-diploid life cycle, with free-living diploid tetrasporophytes and free-living haploid gametophytes, which are morphologically indistinguishable unless they are reproductive (Fig. S1; Heiser et al. Reference Heiser, Amsler, McClintock, Shilling and Baker2020). Meiosis occurs on the tetrasporophytes, resulting in the release of haploid tetraspores. Tetraspores germinate and develop into male and female gametophytes. Gametes are mitotically produced by the gametophytes, but, following fertilization, the zygote is retained on the female gametophyte, where the carposporophyte develops. Each diploid carpospore can germinate into a tetrasporophyte. In natural populations, many thalli are vegetative, rendering it difficult to distinguish the stages. This life cycle results in unique eco-evolutionary consequences that challenge traditional understanding and the utility of common proxies to describe patterns of reproductive system variation (Krueger-Hadfield et al. Reference Krueger-Hadfield, Guillemin, Destombe, Valero and Stoeckel2021). For example, Plocamium sp. has separate sexes, but this does not preclude selfing (intergametophytic selfing; see Klekowski Reference Klekowski1969). Separate sexes, therefore, cannot be used as a proxy to deduce outcrossing in natural populations. Instead, we must use population genetic tools to empirically quantify the relative rates of selfing, outcrossing and asexual reproduction in natural populations.

We developed microsatellites to quantify patterns of genetic diversity and gene flow in Plocamium sp. (Heiser Reference Heiser2022). We chose microsatellites over other approaches for several reasons: 1) microsatellites facilitate the iterative addition of new samples to a dataset, something that is not possible in most genotyping by sequencing (GBS) approaches to identify single nucleotide polymorphisms; 2) microsatellites are an appropriate tool when existing data on ploidy and the reproductive system are absent, which may complicate downstream bioinformatics in GBS approaches; and 3) microsatellites are a powerful tool with which to quantify reproductive mode variation in macroalgae (Krueger-Hadfield et al. Reference Krueger-Hadfield, Guillemin, Destombe, Valero and Stoeckel2021). We collected Plocamium sp. thalli during summers between 2016 and 2018 at ‘East Litchfield’ and Laggard Island near Palmer Station on Anvers Island (see Supplemental Materials for details). All gametophytes had one allele and all tetrasporophytes had one or two alleles, confirming that our 10 polymorphic microsatellite loci are in single-locus genetic determinism (Table S1). There were discrepancies between the direct estimates of null allele frequencies from non-amplification in the haploid gametophytes (< 5%) and those estimated using maximum likelihood in the diploid tetrasporophytes (0–39%; Table S2). When populations are not mating at random, which is an assumption of null allele frequency estimators in diploids, discrepancies between direct and maximum likelihood estimates have been found in other haploid-diploid macroalgae (e.g. Krueger-Hadfield et al. Reference Krueger-Hadfield, Roze, Mauger and Valero2013). As there was also no evidence for short allele dominance (Table S3), these 10 loci are promising for future population genetic analyses.

We performed some preliminary analyses from thalli sampled at ‘East Litchfield’ and Laggard Island (see Supplemental Materials). Thalli at ‘East Litchfield’ appear to be slightly tetrasporophyte biased (Table I). We did encounter several repeated multilocus genotypes (MLGs) in the gametophytes and tetrasporophytes at each site. Most of the repeated MLGs were considered as distinct individuals (or genets) based on P_sex (see discussion in Arnaud-Haond et al. Reference Arnaud-Haond, Duarte, Alberto and Serrão2007). The one exception was a pair of repeated tetrasporophytic MLGs at Laggard Island that we considered as ramets of the same genet. The subtidal environment is highly dynamic, with regular iceberg scour that removes benthic organisms. Plocamium sp. can form secondary attachments (Heiser Reference Heiser2022), making potential fragmentation and reattachment after removal possible. It may also be that we detected a genotypic signature of the carposporophyte in which morphologically distinct, tetrasporophytic MLGs share the same genotype because they originate from carpospores produced in the same carposporophyte or from carpospores that are produced by different fertilization events by the same male-female pair. Engel et al. (Reference Engel, Destombe and Valero2004) and Krueger-Hadfield et al. (Reference Krueger-Hadfield, Collén, Daguin-Thiébaut and Valero2011, Reference Krueger-Hadfield, Roze, Mauger and Valero2013) did not find these genotypic signatures of the carposporophyte in natural populations of Gracilaria gracilis or Chondrus crispus, respectively, suggesting that carpospores might be dispersed in clumps or the genotypic signature of the carposporophyte is localized to a few centimetres. As all carpospores generated from each zygote are genetically identical (barring mutation), we would be unable to detect whether an adult tetrasporophyte originated from one carpospore or hundreds of carpospores.

Table I. Summary statistics for 10 polymorphic microsatellite loci developed in the Antarctic Plocamium sp. and analysed in the gametophytic and tetrasporophytic subpopulations at two sites along the western Antarctic Peninsula. Standard errors are provided for A_E, P_A, H_E and H_O and in parentheses the variance is provided for F_IS.

*P < 0.0025 (with P adjusted to 0.0025 for significance), ***P < 0.001.

¹ For gametophytes, $H_E^A $, unbiased expected heterozygosity was adjusted by a factor of (2N - 1)/(2N - 2); see Engel et al. (Reference Engel, Destombe and Valero2004).

P_HD = ploidy diversity; N = number of thalli; R = genotypic richness; $\bar{r}_d$ = linkage disequilibrium; A_E and P_A = mean and private allelic richness, respectively (using rarefaction and the smallest sample size in gametophytes, N = 9); H_E = unbiased expected heterozygosity; H_O = observed heterozygosity calculated in tetrasporophytes only; F_IS = inbreeding coefficient calculated in tetrasporophytes only (single-locus values are provided in Table S5).

Future explorations of Plocamium sp. populations along the WAP will enable us to determine whether the trend of tetrasporophytic bias with strong heterozygote deficiency, such as that seen at Laggard Island, is common. In other red macroalgae, heterozygote deficiency (e.g. inbreeding coefficient (F_IS) > 0; Table I, and see Table S5 for single-locus values) and high selfing rates have been associated with gametophytic bias (Krueger-Hadfield et al. Reference Krueger-Hadfield, Roze, Mauger and Valero2013). By contrast, tetrasporophytic bias has been associated with outcrossing (F_IS = 0; Engel et al. Reference Engel, Destombe and Valero2004) or with heterozygote excess and clonality (e.g. F_IS < 0; Krueger-Hadfield et al. Reference Krueger-Hadfield, Kollars, Byers, Greig, Hammann and Murray2016). We did detect two ‘fixed‘ homozygous tetrasporophytes (thalli were reproductive at the time of sampling) at ‘East Litchfield’ and four at Laggard Island, suggesting high selfing rates (see Krueger-Hadfield et al. Reference Krueger-Hadfield, Roze, Mauger and Valero2013). However, we will need to use spatially explicit sampling to determine whether the observed heterozygote deficiency is due to the reproductive system and/or a Wahlund effect (see also Heiser Reference Heiser2022).

Haploid-diploid life cycles are predicted to be correlated with selfing, asexual reproduction or both (Otto & Marks Reference Otto and Marks1996). Reduced inbreeding depression is thought to ease transitions to selfing in angiosperms (Barrett Reference Barrett2002) and has been shown to occur at range edges where selfing may be common (Pujol et al. Reference Pujol, Zhou, Sanchez Vilas and Pannell2009). Similar reductions in inbreeding depression may occur in haploid-diploid algae, but the long-lived gametophytes may enable efficient purging of deleterious mutations. The purging of deleterious alleles may further reduce the negative consequences of selfing in natural populations (see Charlesworth & Charlesworth Reference Charlesworth and Charlesworth1987). To date, very little work has explored the effects of inbreeding depression in macroalgae (but see Barner et al. Reference Barner, Pfister and Wootton2011). Future work using these loci in Plocamium sp. will allow us to quantify selfing rates in populations along the WAP, setting the stage for empirical work on the role of inbreeding depression and its evolutionary consequences in haploid-diploid taxa. As the reproductive system partitions genetic diversity within and among populations, understanding patterns of reproductive system variation is critical to deciphering the processes that structure algal-dominated communities.