Eco-genomic analysis uncovers precision-conservation targets for the western Pacific’s southernmost salmonid

Reviewer #1 (Public review):

Summary:

This is an interesting paper on an important topic, the taxonomic and conservation status of some unusual salmonid populations in Taiwan.

Strengths:

The first part of the manuscript is quite strong: the authors sequence and build a reference genome and conduct a phylogenomic analysis. They examine chromosome structure and rearrangements, test for loss-of-function mutations, and do a proteomic analysis. As a stand-alone, this could serve as its own manuscript, perhaps for a more specialized journal.

Weaknesses:

I find this manuscript rather disjointed. The first part of the manuscript is related to phylogenomics of the taxon in question, compared to other nearby species from Japan. The authors go on to describe chromosomal rearrangements, sex-chromosome location, and proteomics. All of these fit within a paper about taxon-level issues. I do find the proteomic analysis perhaps unnecessary. I'm not sure we learn much of substance through this analysis, which is highly speculative.

PSMC analysis seems highly questionable for taxa with such strong genetic structure. If historical Ne and past changes in structure are confounded, what does this analysis provide? I recommend deletion of the analysis included in Figure 1e.

The second portion of the manuscript deals with population structure of three O. formosanus populations, based on RADSeq data. This part reads as a separate manuscript, in my opinion. I think the authors are trying to squeeze too much into one manuscript.

For the second part on population genomics, not enough detail is provided to evaluate the methods, results, and interpretations. For example, not enough detail is provided about each of the three Taiwan populations, the stocking history, and the demographic data collection. The only information available is a brief paragraph in the introduction. Was the Luoyewei (L) population stocked from a brook derived from this population or from Qijiawan (Q)? Why do three L fish have such different levels of MLH? Are these stocked from somewhere else? Are the rest of the fish from one pool, and maybe one family (this would also explain the extremely low contemporary Ne)? Only 17 fish were examined from L, and apparently from one site in the stream; more detail is needed. Are L, Q, and H currently isolated? What is the stocking history? The authors conclude that the Hehuan (H) population has more genetic variation and is likely the result of an unknown native population that bred with stocked fish (which arise from Q). This story does align with the genetic results, but again, more detail is needed. Are there alternative explanations? A more careful treatment would be helpful.

The demographic modeling is not convincing. Not enough detail is provided, and the lack of individual identification of fish makes it so the modeling is very general. It is hard to place too much stock in these vital rate estimates. The methods were fishing, snorkeling, and some electrofishing. Scales were used for ageing, and catch curve analysis was employed. Overall, this is an underdeveloped portion of the paper that is important, but not convincing as written.

Reviewer #2 (Public review):

Summary:

Lee et al. is a comprehensive conservation genomics study that combines a chromosome-level genome assembly (sex-specific, too), population resequencing, coalescent species delimitation, and simulations to reassess the evolutionary status and conservation outlook of the Formosan landlocked salmon, Oncorhynchus formosanus. The authors showed a distinctive genome structure, replete with chromosome fusions and an unusual placement of the sex-determining gene sdY. Across sampling sites, they observed variable levels of genetic diversity, but in a way that was surprising given previous census numbers and conservation history. In particular, the authors report a previously unrecognised native population in Hehuan Creek, and conclude that Hehuan is more resilient to typhoon disturbance than the long-protected Qijiawan population - motivating stream-specific rather than range-wide conservation.

Overall, this is a well-written paper that combines a number of elements that are timely and relevant. It uses state-of-the-art techniques to reach its conclusions and is generally performed to a high standard. It describes a critically endangered species that poses its unique conservation challenges. There are a number of things to like, as well as some substantial shortcomings in this paper.

Strengths:

The genomic resource is excellent. The assembly is well validated (97.3% anchored to 25 scaffolds, 95.7% BUSCO), and the authors generated a separate male assembly specifically to resolve the sex-determining region, allowing XY-shared and Y-specific contigs to be distinguished on coverage rather than inference. This is truly well done, and at a high standard. The synteny evidence for telomere-to-telomere fusions involving at least 14 ancestral chromosomes, against two in O. m. masou, is convincing.

The Hehuan Creek result is the paper's most valuable contribution. Elevated heterozygosity, short and infrequent runs of homozygosity, and private alleles absent from the Qijiawan broodstock are difficult to reconcile with a purely reintroduced origin. The contrast with Luoyewei is a clean and useful cautionary case for hatchery supplementation.

Weaknesses:

(1) The species-rank claim is featured in the abstract, but it is made with any level of rigour in the paper. "New species" appears once, in the abstract (l. 32). The Results conclude only that O. formosanus is a distinct evolutionarily significant unit (ll. 188-191), which itself can be well-justified, but it's far from a taxonomic rank (see author's own ref 10). No species concept is explicitly named anywhere, and the taxon is referred to across the manuscript as a subspecies (l. 68), a "new species" (l. 32), and an ESU (l. 189) in turn.

(2) Gene flow is asserted, not tested, and two divergence estimates disagree twentyfold. The abstract reports "no detectable gene flow for ~50,000 years." That figure is a divergence time from BPP under the A00 model, which contains no migration parameter; a model that cannot fit gene flow cannot report its absence. Separately, Figure 1b shows a split at 1.15-5.09 Mya (Figure 1b), while the ddRAD coalescent places the same split at ~50 kya (Figure 1d). The explanation offered (ll. 417-421, "differing temporal sensitivity of genomic markers") is not a mechanism.

(3) The placement of sdY is unresolved, and the paper's own figures conflict with its text. Figure S10 and Table S6 both make O. formosanus chr13 homologous to O. m. masou chr32, whereas reference 28 - on which the authors rely - places the sdY contig on O. m. masou chr7, whose O. formosanus homologue is chr5 (Table S6). These cannot both be correct, and Figure S10's caption compounds the confusion by attributing chr13 to masou and omitting the chr32 track entirely.

(4) The population-viability model's stated mechanisms are contradicted by the authors' own supplementary tables. The Discussion attributes Qijiawan's vulnerability to "lower juvenile survival, decreased fecundity, and narrower terminal age class representation" (ll. 522-525). Table S10 gives Qijiawan higher age-0 survival (0.202/0.616 vs 0.184/0.615); Table S11 gives it higher fecundity at every reproductive age (7.68/19.27/7.86 vs 6.10/8.95/4.14); Table S9 gives it a broader terminal age class (5.0% vs 0.8% age-3 in November). The only parameter favouring Hehuan is age-1 survival - 0.087 (95% CI 0.000-0.180) versus 0.131 (0.093-0.187) under typhoon, and 0.054 (0.000-0.167) versus 0.087 (0.047-0.149) at baseline. Both Qijiawan intervals include zero and overlap Hehuan's, yet a reported extinction odds ratio of 4.48 rests on this difference.

(5) The two streams were not measured equivalently, and every asymmetry favours the conclusion.