﻿On the identity of Thymushumifususvar.aureopunctatus (Lamiaceae) and taxonomic notes on the Th.richardii complex

﻿Abstract The name Thymushumifususvar.aureopunctatus, described from Bosnia and Herzegovina, is lectotypified, and its taxonomic value is discussed. Thymusrichardiisubsp.richardii is currently considered an endemic subspecies common to Mallorca (Balearic Islands) and Bosnia and Herzegovina from the Balkan Peninsula. Specimens identified as Th.richardii from both Balearic Islands and Bosnia and Herzegovina were studied to determine if they are indeed the same taxonomic entity. Detailed micromorphological observations and morphometric analysis, suggest that the Balkan plants (Th.humifususvar.aureopunctatus) and the Majorcan populations (Th.richardiisubsp.richardii) are clearly separate entities. For the former name, based on morphological, phytochemical, biogeographical and present results, we propose the subspecific rank, as Th.richardiisubsp.aureopunctatuscomb. & stat. nov. Full descriptions of all five subspecies currently accepted within Th.richardii are provided.

As currently circumscribed, Th. richardii subsp. richardii presents a striking distribution pattern, since the isolation between both areas (Mallorca and the Bosnia) is remarkable, and there are no other cases of shared endemism between these areas. Furthermore, i) the differences between the habitat occupied by Th. richardii subsp. richardii in both areas, ii) the differences in the composition of essential oils (Llorens et al. 2014), and iii) the fact that the Bosnian and Herzegovinian population was initially recognised as a separate taxon (Th. humifusus var. aureopunctatus Beck) by several authors (Malý 1908(Malý , 1923Ronniger 1930a, b) invites a re-evaluation of the inclusion of the latter taxon within the synonymy of Th. richardii subsp. richardii as originally proposed by Jalas (1971).
In order to elucidate the taxonomic identity of Th. humifusus var. aureopunctatus, we have sampled specimens from the Bosnian and Herzegovinian and Balearic populations of Th. richardii in the field for a detailed comparison. On the other hand, the morphological characters used to separate the rest of the subspecies recognised in Th. richardii have also been analysed in detail. Finally, a multivariate morphometric analysis based on quantitative traits was carried out to clarify the relationships among taxa within the Th. richardii complex.

Plant material
This study is based on analysis of relevant literature, field surveys and examination of herbarium specimens kept in BC, BCN, COI, HBJS, MA, P, PAL, SARA, VAL, ZA, ZAGR (herbarium codes according to Thiers 2021) and the Herbarium of the Balearic Islands University. For the typification purposes, herbarium specimens deposited at BC, BM, L, P, and PRC were studied using the online herbarium databases or images were requested.
Furthermore, the plant material of recently collected samples of Th. humifusus var. aureopunctatus and Th. richardii subsp. richardii from Bosnia and Herzegovina and Mallorca was analysed, too (Fig. 1A). In total, 61 individuals from seven populations (see Appendix 1) were surveyed for micromorphology and quantitative morphometry.
Morphological characters recognised as taxonomically discriminant within the Th. richardii complex (Jalas 1972;Morales 2010; and our own observations) were scored either in the field or in the herbarium specimens (Table 1, 3). Morphological observations of materials were carried out under a Zeiss Stemi DV4 binocular stereoscopic microscope. We scored both qualitative and quantitative traits in evaluation of taxa, the latter ones were used to describe the pattern of morphological variation and relationships among taxa.
Micromorphology was observed on calyces, which were glued directly to aluminium stubs, coated with 40-50 nm gold, and examined with a scanning electron microscopy (SEM) (Zeiss Merlin FE-SEM) at 5 kV.

Data analysis
Descriptive statistics (mean, minimum and maximum value, standard deviation and coefficient of variation for each of the studied characters at the taxon level) and univariate statistics (one-way ANOVA followed by Tukey's test) were calculated to test the significance of differences between taxa within the complex. Overall morphological variation of quantitative traits and relationships of the sampled taxa was evaluated using Principal Component Analysis (PCA). Thymus richardii subsp. vigoi was excluded from analysis due to distinct characters in relation to the other taxa (see Identification key). Means of averaged and standardised values of individuals were used as a matrix data in PCA. PCA was computed on the correlation matrix data of all scored traits. The axes with Eigen values > 1 were used in analysis. PCA computation, descriptive and univariate statistics were run in PAST ver. 3.14 (Hammer 2016).

Results and discussion
The variation based on SEM micromorphology, univariate and multivariate morphometrics (PCA) of taxa included within Th. richardii complex is described and their taxonomic value of the characters is here discussed.

Variation of individual quantitative morphological traits within the Th. richardii complex
Mean values of the analysed traits go in favour of morphological differentiation among taxa (Table 1). In general, coefficients of variation (CV) did not exceed 20% (Table 1). The values in Th. richardii subsp. vigoi should be treated with caution because their calculations were based on three individuals. In most cases the coefficients of variation had low (CVs ≤ 10%, 29 cases) and moderate values (CVs from 10 to 20%, 36 cases) ( Table 1). High values of coefficients of variation were observed for the trait length of longer cilia of upper calyx teeth (LCU) in each taxon and for the trait longer inflorescence length (IL). The traits calyx length (CL) and calyx tube length (CTL) showed the lowest values of coefficients of variation (Table 1).
One-way ANOVA displayed significant differences between mean values of quantitative traits for all subspecies (p ≤ 0.01). The Tukey's test revealed significant differences among subspecies for the most of the studied traits (     (Fig. 1C). Following characters (CL, LTL, CTL, and BL) with moderate coefficients of correlations were associated with PC1 (Table 3). The characters LEH, LCL and LCU contributed to the PC2. The highest correlations with the PC3 showed derived trait L/W and leaf width (LW) which contributed to the separation of Th. richardii subsp. nitidus (Table 3). Principal component analysis of quantitative morphological data demonstrates that allopatric populations of the Th. richardii complex are clearly distinguishable according to their taxonomic affiliation. Variation in particular morphological traits indicated a similar pattern observed in PCA, confirming a high level of morphological differentiation between the studied taxa. High levels of both morphological and genetic differentiation within plant complexes are not surprising in the Mediterranean. This pattern of variation, which often results in endemism, is particularly pronounced for populations inhabiting the Mediterranean islands (Thompson 2020). Due to different geological and biogeographical processes, long-term isolation, adaptation and specialization to contrasting habitats, the ancestral populations of Th. richardii diverged into distinct entities across the Mediterranean and the Balkans.

General habit
All the taxa included within the Thymus richardii complex are woody perennials with young or flowering stems with hairs on all faces, more or less evenly distributed. These hairs are eglandular, usually retrorse, up to 0.2 mm long (0.4 mm long in Th. richardii subsp. ebusitanus), intermixed with sessile glands. According to Morales (2010), the plant length separates the populations from Mallorca from those of Ibiza (7-13 cm vs. 10-24 cm, respectively), but in Table 2. The studied traits differentiating between taxa based on result of the Tukey's t tests (p = 0.01) (abbreviations are as in Table 3).

Leaves
All the studied taxa have flat leaves, not ciliate at base, with entire margins, except in Th. richardii subsp. vigoi, which has denticulate leaves. On the basis of leaf morphology (Riera et al. 2007) Th. richardii subsp. vigoi is easily separable from the rest of the members of the Th. richardii complex. Leaves shape varies from ovate to elliptical. Jalas (1972) attributed to Th. richardii subsp. nitidus leaves more than twice as long as wide. Certainly Th. richardii subsp. nitidus usually has leaves with a higher length / width ratio than the rest of the taxa (Table 1), but we have studied plants of the island of Marettimo with leaves less than twice as long as wide. On the other hand, some Majorcan specimens of Th. richardii subsp. richardii have leaves more than twice as long as wide.
The leaves have spheroidal yellowish-reddish glands, and sometimes scattered hairs exist in several taxa of this complex. Some specimens of Th. richardii subsp. ebusitanus, Th. richardii subsp. vigoi and Th. richardii subsp. nitidus have a hairy main midrib in its basal half; this hairiness sometimes extending towards adjacent areas of the blade. Nevertheless, this character seems not to be sufficiently constant for taxonomic purposes.

Inflorescence
Flowers are arranged in distinct inflorescences, usually capitate to more or less elongate (up to 62 mm long in Th. richardii subsp. ebusitanus, Table 1). Bracts are similar to leaves, but smaller, and the bracteoles linear to linear-lanceolate. Pedicels are somewhat longer than documented for the species (Morales 2010), since in Majorcan plants of Th. richardii subsp. richardii can reach up to 5 mm long.

Calyx
Upper calyx-teeth are conspicuously different from lower. The upper lip teeth are usually narrower in Th. richardii subsp. vigoi. The calyx is green to purplish-green or to purple-violet. This colour variation can be observed within the same population, and the purplish coloration usually occurs in specimens that grow in more exposed places.
Regarding the calyx length, Th. humifusus var. aureopunctatus shows the lowest values, whereas the longest are those of the Majorcan populations of Th. richardii subsp. richardii (Table 1; Fig. 2). On the other hand, the length of the lower teeth of the calyx also allows separating the previous taxa (Table 1, 2). The presence of shorter calyces in Th. humifusus var. aureopunctatus was documented by Morales (2010), but so far this variation had not been quantified. From our point of view, the calyx length is a diagnostic character to separate the Balkan and the Balearic populations, together with other morphological characters (Table 1, 2).
The calyces are more or less hairy, with spheroidal yellowish-reddish glands. Our results show that the characters related to the hairiness of the calyx have taxonomic relevance in the Th. richardii complex. Calyx indumentum in Th. richardii subsp. ebusitanus is dense, with long eglandular hairs (up to 1 mm long), mainly on the margins of the lower teeth of the calyx and the ventral part of the calyx tube (Figs 2, 3). On the contrary, the calyx in the Majorcan populations of Th. richardii subsp. richardii is glabrescent (the upper lip and the dorsal surface of the calyx tube are glabrous or glabrescent) and the hairs are much shorter (Table 1). Thymus humifusus var. aureopunctatus has glabrescent to sparsely hairy calyces, but the eglandular hairs are usually more abundant and longer than in the Majorcan plants of Th. richardii subsp. richardii.
Stipitate glandular hairs are found in calyces (tube, teeth and even on the adaxial surface of upper teeth) of several taxa (Table 1). As noted by Jalas (1972) these glandular hairs are particularly abundant in Th. richardii subsp. nitidus (Figs 2, 3). However, stipitate glandular hairs are also usually found (in variable density) in Th. richardii subsp. ebusitanus, while in Majorca the specimens having these glandular hairs are rather rare but are observed on specimens from Coma de n'Arbona (BC 651145). These glandular hairs were not documented for Balearic plants of Th. richardii by Morales (2010). This character seems to be variable in the Balearic populations, since in the same locality there are plants without these glandular hairs.

Corolla
The upper lip is emarginate and the lower has 3 subequal lobes (middle lobe somewhat longer). The corolla is more or less hairy on the outer surface, with spheroidal yellowish-reddish glands. Its colour varies from pale rose (sometimes whitish or cream in Th. richardii subsp. vigoi) to pinkish-purple. The coloration is somewhat variable within the different taxa and in our opinion has no taxonomic significance.

Taxonomic treatment
The Majorcan and the Balkan populations, which were included within typical T. richardii (Jalas 1971(Jalas , 1972Morales 2010) are morphologically distinct; they differ in several characters including calyx size, lower calyx teeth length, length of hairs on the calyx tube, length of pectinate hairs of lower calyx teeth and indumentum density on the calyx (Figs 1-3; Table 1, 3). The Majorcan plants have, compared to those from Bosnia and Herzegovina, longer and less hairy calyces, with shorter hairs and longer lower calyx teeth with shorter (and less dense) pectinate hairs. Examination of herbarium specimens from five populations (16 specimens from Mallorca, 21 from Bosnia and Herzegovina) plus other specimens (see additional specimens examined) revealed that the diagnostic characters are constant within each geographic group. The morphological and biochemical (Llorens et al. 2014) differentiation between the Majorcan and the Balkan populations and their allopatric distribution (they are separated by a gap of ca. 1.300 km) firmly support the recognition of two subspecies, since the level of morphological differentiation between the two taxa does not meet the criteria commonly used to delimit species in Thymus. Certainly, further research using molecular markers is needed to reveal genetic relationships and biogeographic history of the Th. richardii complex. Type. Holotype (see Rosselló and Sáez 2001: 109): P-Lamarck. Description. Stems up to 47 cm long, procumbent to reptant. Leaf blade up to 13 × 7.7 mm, broadly ovate to elliptical, entire. Inflorescence 15-30 mm long, capitate to oblong; bracts up to 11 × 7.8 mm, similar to leaves, entire, glabrous. Calyx 6-8 mm long, glabrescent (sometimes glabrous), with eglandular hairs up to 0.3 mm long, occasionally with scattered stipitate glandular hairs; calyx tube 2.2-3.2 mm long, glabrescent (sometimes glabrous on the dorsal surface), with eglandular hairs up to 0.3 mm long on the ventral surface; central tooth of upper lip 1.3-2.2 mm long, lower teeth 2.8-3.8 mm long, with pectinate hairs up to 0.3 mm long. Corolla 7-11 mm long, rose to pinkish-purple (Fig. 4, C, E).
Remarks. This is a rare plant, documented from three localities in the north of Mallorca (Ternelles mountain, Formentor peninsula and Puig Major) of which we have only been able to verify its presence in the last locality, growing on cliffs with very difficult access. This taxon could be facing a population decline. Bianor (1917) at the beginning of the 20 th century, considered it as abundant in the Puig Major ["Abondant dans les endroits peu accessibles"]. In fact, there are dozens of specimens from this mountain and which are preserved in various herbaria; mostly collected in the late 19 th and early 20 th centuries. Currently, Th. richardii subsp. richardii is very scarce at the same locality where it was reported by Bianor (1917) and the plants are practically inaccessible if climbing techniques are not used. Another population located on a different slope of the same massif is also scarce and very difficult to access. This possible population decline could be due to a loss of potential habitat and intense predation by feral goats (Capra hircus).    Fig. 5).
Habitat. Sandy dolomites and dolomitic rocky places, 400-1040 m a.s.l. Remarks. Beck (1887) described Th. humifusus var. aureopunctatus from "In saxosis prope Konjicam" [Bosnia and Herzegovina] and related this new variety to Thymus humifusus Bernh. ex Link, which is currently regarded a synonym of the tetraploid Th. praecox Opiz (Jalas 1971;Euro+Med 2006;Plant List 2021;WFO 2021). Günther Beck (1856Beck ( -1931 was a Bohemian botanist, and his herbarium is currently kept at PRC and W (Stafleu and Cowan 1976). We have been able to locate original material of Th. humifusus var. aureopunctatus at PRC. This is a well-prepared specimen; it matches the description and the provenance indicated in the protologue. Therefore, we designate the specimen with barcode PRC 455886 as the lectotype of the name Th. humifusus var. aureopunctatus (Fig. 5) Bartolucci et al. 2013Bartolucci et al. : 1310. Description. Stems up to 25 cm long, procumbent or suberect. Leaf blade up to 10 × 4.5 mm, elliptical, entire. Inflorescence 8-30 mm long, subcapitate to oblong; bracts up to 7 × 4 mm, similar to leaves, entire, glabrous to hairy at margin and midrib (eglandular hairs up to 0.4 mm long, mixed with stipitate glandular hairs). Calyx 4.5-6.3 mm long, densely covered by stipitate glandular hairs and sparse eglandular hairs up to 0.5 mm long; calyx tube 1.9-2.5 mm long, with eglandular hairs up to 0.5 mm long on the ventral surface; central tooth of upper lip 0.8-1.5 mm long, lower teeth 2-3 mm long, with pectinate hairs up to 0.5 mm long. Corolla 6.5-9.5 mm long, pale rose.
Chromosome number. Unknown. Distribution. Endemic to Alicante and Valencia provinces (Spain).

Remarks.
Plants which were considered to be hybrids between Th. richardii subsp. vigoi and T. piperella L. have been called T. × bolosii. The hybrid has been reported from a small area of Serra de la Safor, eastern Spain (Riera et al. 2020

Identification key for Thymus richardii complex
We propose the following key for the subspecies of the Thymus richardii complex in order to include the new proposed subspecies.