Melichrus urceolatus

Melichrus urceolatus View in CoL and M. erubescens

In the genus-wide phenetic analysis, the Melichrus urceolatus segregates (see Table 1 for summary of OTUs) clustered in five separate groups, spread across the ordination space and phenogram ( Fig. 3). Strikingly, a very similar pattern of variation was returned in a genus-wide PCA and SplitsTree analysis of the DArTseq dataset ( Fig. 7), where the M. urceolatus segregates also clustered in five highly distinct clusters but with some variation in which OTUs grouped together. These results demonstrated that M. urceolatus has been used as a ‘catch all’ species requiring significant taxonomic restructuring.

Both the morphological and molecular analyses demonstrated that Melichrus erubescens and M. urceolatus are highly distinct from each other, despite decades of confusion regarding the boundary. The analyses also showed that some OTUs segregated from M. urceolatus , such as M. sp. Galambary and M. sp. Gardens of Stone, had more in

(a) (b)

30°S

35°S

common with M. erubescens genetically, morphologically or in some cases both. This explains why the status quo taxonomic boundary between M. urceolatus and M. erubescens has been so difficult to apply in practice.

Across all analyses, Melichrus adpressus sens. str. was supported as distinct from M. urceolatus , with which this was synonymised by Bentham; distinct from M. adpressus sensu Paterson , to which the name was misapplied by Mueller and Paterson; and distinct from M. erubescens sensu Paterson , with which many of the populations have been confused in herbarium collections. The results for M. adpressus sens. str. corroborate the recognition as a unique species, as originally proposed by Cunningham and formalised by De Candolle in 1839. As such, the name M. adpressus should be restored to the original application, sensu De Candolle (1839).

On the balance of evidence, we recommend that the OTUs Melichrus sp. Galambary and M. sp. Kaputar be synonymised with M. adpressus sens. str. Morphological and molecular evidence indicated a close relationship between M. sp. Galambary and M. adpressus sens. str. across all analyses. There was some indication of an approximately north-south disjunction in morphology and genetics among the populations examined of M. sp. Galambary. This observation requires targeted sampling and further testing before accepting this phrase name as a separate species. Melichrus sp. Kaputar, known only from a single, isolated outcrop on Mount Kaputar, New South Wales is morphologically consistent with M. adpressus sens. str. Molecular analyses on the other hand, suggested that the OTU is related to a lineage containing M. adpressus sens. str. but is genetically divergent. This was particularly evident in

–0.75 –0.50 –0.25 0.00 0.25 M. procumbens

M. sp. Torrington

M. medius

M. urceolatus

M. sp. Silent Grove M. sp. Tara

M. sp. Isla Gorge

M. sp. Mareeba Populations

M. sp. sp. Galambary

M. sp. Gilgandra M. erubescens

M. sp. Kaputar M. sp. Gardens of Stone

M. adpressus sens. str.

M. sp. Yuraygir

M. sp. Boonoo Boonoo M. adpressus s. Paterson

M. sp. Gurulmundi

M. sp. Kroombit Tops M. sp. Herberton

M. hirsutus M. gibberagee

He Ho Fis the PCA and SplitsTree analysis. However, the results of the STRUCTURE analysis were more equivocal and the conStruct analysis suggested that this was only minimally differentiated from M. adpressus sens. str. Mount Kaputar is a known area of endemism (New South Wales Department of Planning, Industry and Environment 2021) the uniqueness of M. sp. Kaputar contributes to the conservation value of the area. However, in the absence of diagnostic characters to distinguish M. sp. Kaputar reliably from M. adpressus sens. str., we recommend that this be synonymised.

Melichrus sp. Gilgandra is strongly morphologically distinct from M. adpressus View in CoL sens. str. but only weakly genetically diverged. This putative species with urceolate flowers and small, broad, often recurved leaves had more in common in the phenetic analyses with M. sp. Inglewood and M. urceolatus View in CoL than M. adpressus View in CoL sens. str. ( Fig. 4 View Fig ). However M. sp. Gilgandra does share some morphological characters, such as fruit texture and calyx length, with M. adpressus View in CoL sens. str. ( Table 4c). The combination of strongly glaucous, blue-coloured leaves, pink petiole margins, urceolate flowers and wrinkly fruit epidermis make this putative species highly recognisable. The strong morphological but weak genetic discontinuity between M. sp. Gilgandra and M. adpressus View in CoL sens. str. could indicate that M. sp. Gilgandra has only relatively recently diverged from a lineage containing M. adpressus View in CoL sens. str. and is yet to accumulate many differences in allele frequency through genetic drift. The area around Gilgandra in New South Wales where this putative entity occurs is at the more arid end of the environmental envelope in which M. adpressus View in CoL sens. str. occurs, therefore some of the morphological differences seen in M. sp. Gilgandra, such as smaller, glaucous leaves could be evidence of local selective adaptation ( Solbrig 1994). These may also represent a plastic response to the environmental conditions ( Carvajal et al. 2017). Ideally, this would be directly tested through greenhouse experiments (see Collins et al. 2022) but as species of Melichrus View in CoL are challenging to cultivate from seed and cuttings, this has not been feasible.

As explained in the Introduction, to satisfy requirements for species status under the unified species concept, evidence should indicate that M. sp. Gilgandra is an independently evolving metapopulation lineage ( de Queiroz 2007), identifiable by corresponding discontinuities in a variety of data sources. The evidence from morphological analyses

Systematic Botany 38 (2025) SB24031 (Continued on next page)

31 Systematic Botany 38 (2025) SB24031 supports M. sp. Gilgandra as a distinct species but the molecular data suggests that this is a morphologically distinct population of M. adpressus sens. str. Collections of new populations and data or greenhouse experiments may tell us more about the evolutionary trajectory of M. sp. Gilgandra. Until further evidence becomes available, we recommend that this be described as a subspecies of M. adpressus sens. str. Recognition as a subspecies will allow formal description of the unique morphology of this OTU. This is important for identification purposes and communicating the diversity value to conservationists.

A specimen of Melichrus sp. Gardens of Stone collected by Erwin Gauba near ‘Pigeon House Mountains’ (CANB 003865) was listed in Paterson (1958) under M. urceolatus but in both the molecular and morphological analyses all samples of M. sp. Gardens of Stone clustered closer to M. erubescens . More detailed analyses showed this OTU to be similar to, but diagnosable both morphologically and genetically as distinct from M. erubescens . We therefore recommend that this be described as a new species.

Melichrus sp. Silent Grove clustered near to, but distinct from M. erubescens in the morphological analyses. However, PCA and SplitsTree genetic analysis demonstrated that M. sp. Silent Grove is more genetically similar to, yet distinct from, M. urceolatus . Melichrus sp. Silent Grove does share some morphological traits with M. urceolatus , particularly the surface texture of the dried fruits but is easily distinguished by several characters, especially the adpressed and straight young leaves ( v. young leaves erect to strongly spreading, often recurved) and the absence of a white margin on leaves ( v. leaves with a white margin ~ 1 mm wide). Given the distinctiveness of M. sp. Silent Grove across analyses, we recommend recognising this narrow range endemic as a new species. The results of the STRUCTURE analysis support this recommendation but raise some questions regarding the evolutionary history of this new species. In the STRUCTURE analysis, M. sp. Silent Grove was represented almost entirely by a single ancestral population. This ancestral population was shared in part by all other geographically proximal Melichrus species, including M. sp. Torrington, M. urceolatus and M. sp. Boonoo Boonoo. There are plausible mechanistic explanations for such a pattern of admixture including that northern New South Wales is an evolutionary centre of diversity for this genus and that this layer represents an old, widely shared ancestral population. This pattern could also be an artefact of the analysis, as M. sp. Silent Grove was only represented in analyses by the single known population and thus warrants further testing.

Melichrus sp. Inglewood grouped with M. urceolatus in all genetic analyses. In morphological analyses, this clustered close to M. urceolatus but was separated by the particularly small leaves. No further characters were found to reliably segregate this putative species from all M. urceolatus populations. We recommend this be treated as a synonym of M. urceolatus .

A northern cluster was detected among the Melichrus urceolatus samples across all analyses. conStruct analysis indicated that the discontinuity observed in the STRUCTURE results was likely substantially independent of the influence of isolation by distance. Despite the strong genetic discontinuity between the northern cluster and remaining M. urceolatus samples, there was no morphological evidence to support species delimitation. Similarly, STRUCTURE results indicated substantial divergence and interesting patterns of admixture between populations of M. medius and some M. urceolatus . This genetic divergence was not matched by a corresponding discontinuity in morphological characteristics sufficient to recommend description as a separate species. The continued synonymy of M. medius with M. urceolatus is recommended.

Molecular data decisively placed Melichrus sp. Tara as conspecific with M. sp. Isla Gorge, and together, both were highly disjunct from M. urceolatus , despite being previously included under a broader concept of this species by Paterson (1958). Morphological analyses also strongly supported M. sp. Isla Gorge plus M. sp. Tara as distinct from M. urceolatus and we recommend that the two be described as a single new species.

Molecular and morphological results supported Melichrus sp. Mareeba as a species distinct from M. urceolatus . Furthermore, molecular results indicated that M. sp. Mareeba may have evolved from a hybrid or allopolyploid ancestor(s), where the closest living relatives of the ancestral parental lineages are M. sp. Isla Gorge plus M. sp. Tara and M. sp. Herberton plus M. sp. Kroombit Tops. This result is most strikingly apparent in the admixture pattern observed in the SplitsTree and STRUCTURE analyses ( Figs. 7, 11 View Fig ). The elevated HE and HO of these populations, and the extremely negative FIS are more consistent with an allopolyploid or possibly a clonal F1 diploid hybrid ( Fig. 13 View Fig ). A sexual diploid hybrid can likely be ruled out, as a single generation of outcrossing would be expected to bring FIS much closer to zero, though chromosome counts are needed to understand the possible involvement of polyploidy. Morphological phenetic analyses weakly separated M. sp. Mareeba from M. sp. Isla Gorge and M. sp. Tara samples clustered close to M. sp. Mareeba. Melichrus sp. Mareeba shares some morphological characters with M. sp. Herberton, most notably the presence of a wide white leaf margin. Several characters for the decisive diagnosis of M. sp. Mareeba as distinct from M. sp. Isla Gorge plus M. sp. Tara and M. sp. Herberton were identified ( Table 4 b). On the basis of this evidence, we recommend that M. sp. Mareeba be described as a new species. Results of unpublished phylogenetic analyses (Kennedy et al. unpublished manuscript) that interrogate the putative hybrid ancestry of M. sp. Mareeba shed more light on this taxon and bolster evidence supporting the delimitation (see Nauheimer et al. 2021).

The final segregate from Melichrus urceolatus was M. sp. Salvator Rosa but this was unable to be located in the field and thus excluded from the analyses, therefore the status of this segregate remains unresolved.