The Lotus angustissumus Group (Fabaceae): Can Phylogenetic Patterns Be Accommodated by a Taxonomic Concept?

The Lotus angustissimus group represents an example of obvious contradictions between the molecular and morphological data. This group includes from six to eight mostly annual species of Lotus section Lotus, with the center of species diversity in the Mediterranean. We performed a phylogenetic analysis of the genus Lotus with an expanded representation of all known species of the L. angustissimus group using both nuclear (nrITS) and a set of plastid DNA markers and compared the results with traditional taxonomy of this group. Our results demonstrated a non-monophyletic nature of the study group. In addition, the nrITS and plastid dataset tree topologies were incongruent with each other in both Bayesian and ML analyses. We revealed very complex phylogenetic relationships among the species of the group. Important results obtained in this study are: (1) genetic and geographical differentiation in the latitudinal direction (between L. angustissimus and L. praetermissus) and in the longitudinal direction among closely related accessions identified as L. castellanus, L. lourdes-santiagoi and L. palustris; (2) close genetic links between the Anatolian endemic L. macrotrichus and L. praetermissus from Eastern Europe and Central Asia; and (3) the hybrid nature of L. subbiflorus with L. parviflorus as its presumed male parent species. These results are discussed in the context of morphology, biogeography and taxonomy.


Introduction
Molecular phylogenetic studies have radically changed the traditional taxonomy of many groups of flowering plants, including the Fabaceae family (e.g., [1]).In some cases, taxa isolated by classical taxonomy methods have been confirmed using molecular phylogenetic approaches.In other cases, there were obvious contradictions between the molecular and morphological data, and one such example is the Lotus angustissimus group.This is a relatively small group of mostly annual Lotus species of the Lotus section, which includes, according to various points of view, from six to eight species and has a center of species diversity in the Mediterranean.

Taxonomic Composition of the Lotus angustissimus Group
The species Lotus angustissimus L. was described by Linnaeus [2].Heyn included it and a number of morphologically similar species in the Lotus angustissimus group [3].She showed the importance of keel beak and fruit length features for the delimitation of species in this group.Heyn [3] separated species of the group into two subgroups: with a straight keel beak (L.angustissimus, L. macrotrichus Boiss., L. suaveolens Pers.and L. parviflorus Desf.) and a curved keel beak (L.subbiflorus Lag. and L. palustris Willd.).
Kramina conducted a taxonomic revision of the Lotus angustissimus group [6].She demonstrated that the priority name for the species with a long straight keel beak and middle-sized pods is L. subbiflorus [7], not L. suaveolens [8].She also argued why the name L. hispidus Desf.cannot be used for this taxon and demonstrated that the name L. castellanus Boiss.& Reut.has a priority for the species with a long, curved keel beak.She discovered a new significant character, i.e., ovary and pod pilosity along the ventral suture, which distinguishes L. castellanus from L. palustris.In L. castellanus, fruits and ovaries are hairy along the ventral suture and in L. palustris, they are glabrous or sometimes with a few trichomes [6].Multivariate morphological study of the L. angustissimus group from the same work [6] demonstrated the following results: (1) L. praetermissus is not distinguished from L. angustissimus by main diagnostic features (except those of seeds); (2) L. macrotrichus is close to L. angustissimus and L. praetermissus; (3) there are transitional morphological forms between L. castellanus and L. palustris; (4) analysis carried out on a large set of morphological features led to the division of the dataset into four groups-a.L. parviflorus, b.L. subbiflorus, c.L. angustissimus + L. praetermissus + L. macrotrichus, and d.L. castellanus + L. palustris-but for a reduced set of characteristics, including only diagnostic ones, and connections were found between L. subbiflorus and groups a and d.
Pina and Valdés described a new species in the L. angustissimus group, Lotus lourdessantiagoi Pina & Valdés, close to L. castellanus but differing by hairy standard petals.They also clarified a set of diagnostic characteristics for distinguishing species in the group [9].

Chromosome Numbers in the Lotus angustissimus Group
Traditionally, the Lotus angustissimus group was included in Lotus section Lotus, which is characterized by main chromosome number x = 6, in contrast to the other sections of the genus with main chromosome number x = 7 [10].According to literature data, species of L. angustissimus are diploids with 2n = 2x = 12 (L.castellanus, L. parviflorus, L. praetermissus), tetraploids with 2n = 4x = 24 (L.subbiflorus) or have two ploidy levels 2n = 12, 24 (L.angustissimus) or variable chromosome numbers 2n = 12, 14, 24 (L.macrotrichus, L. palustris) [3,[10][11][12][13].The number 2n = 14 indicated for the species of L. angustissimus group is doubtful.In general, the chromosome numbers published for species of the L. angustissimus group should be treated with caution due to the difficulties in determining the species in this group and the different taxonomic views of the authors.

Geographical Distribution of Lotus angustissimus Group
Data on the geographical distribution of the species of the Lotus angustissimus group are summarized from several sources [3,6,14].Lotus angustissimus is distributed in the Mediterranean, Europe (except the northern part), and the Middle East.The distribution area of Lotus praetermissus is in Eastern Europe and Central Asia (Kazakhstan, South Ural and South Siberia).Lotus macrotrichus is endemic to Western Anatolia and adjacent islands.Of a pair of closely related species, Lotus castellanus is common in the Western Mediterranean, and Lotus palustris is sporadically found throughout the Mediterranean and in Western Europe, although the exact range of the latter is unknown due to poorly defined Plants 2024, 13, 101 4 of 22 morphological boundaries between the two species.Lotus lourdes-santiagoi is endemic to Andalusia (southwestern Spain).The main distribution area of Lotus parviflorus and Lotus subbiflorus is the Western Mediterranean and Macaronesia.Lotus subbiflorus also was rarely recorded from the Eastern Mediterranean and southwest England.
Species of the Lotus angustissimus group (especially, L. angustissimus and L. subbiflorus) have spread beyond their main range and formed secondary ranges in Australia, New Zealand, South and North America, South Africa and the Pacific Islands [6,14].

Previous Phylogenetic Studies of Lotus angustissimus Group
Early molecular phylogenetic study of the tribe Loteae investigated phylogenetic relationships among several species of the L. angustissimus group by nrITS markers [15].In this work, the group was not monophyletic: L. angustissimus was clustered with L. parviflorus in a common clade, and L. palustris with L. corniculatus in another unrelated clade.L. subbiflorus took a separate position.The results obtained by Allan et al. [15] were not confirmed by subsequent studies.
Degtjareva et al. [16] analyzed nrITS markers in a representative sample of Lotus species and revealed the separation of the section Lotus into two highly supported clades.The first clade included L. subbiflorus, L. parviflorus and L. conimbricensis, and the second all the other studied species of the section, including L. praetermissus and L. angustissimus, as well as representatives of the groups L. pedunculatus and L. corniculatus.Interestingly, these two clades were not sister to each other on the phylogenetic tree [16].Subsequent phylogenetic studies of the genus Lotus revealed that L. conimbricensis is not related to any species of the Lotus angustissimus group [17,18].
Phylogenetic study of a representative set of Lotus species based on nuclear (nrITS, nrETS) and plastid (psbA-trnH, rps16) DNA markers revealed subdivision of the genus Lotus into the so-called Southern clade and one or more (according to different markers) Northern clades [17].The Southern clade includes the majority of sections of the genus, and the Northern one only three sections (i.e., Lotus, Dorycnium and Bonjeanea).The study demonstrated a non-monophyletic pattern of the section Lotus [17].The species of the Lotus angustissimus group belong to the Northern clade (by plastid markers) or are distributed among the northern clades (by nuclear markers).None of the markers confirmed the monophyly of the Lotus angustissimus group as understood by Heyn [3].Close relationships were demonstrated between L. subbiflorus and L. parviflorus and between L. castellanus and L. palustris by ITS marker, and between L. subbiflorus and L. castellanus and between L. angustissimus and L. praetermissus by plastid markers.The plastid dataset also clearly showed a close relationship between L. angustissimus and L. pedunculatus groups.However, an insufficiently presented sample of species of the Lotus angustissimus group does not allow the authors to draw confident conclusions about the phylogenetic relationships of the species of the group [17].The species L. macrotrichus and L. lourdes-santiagoi have not been studied by molecular methods, and their phylogenetic position was unknown until now.
The present study was aimed at conducting a phylogenetic analysis of the genus Lotus with an expanded representation of all currently known species of the Lotus angustissimus group using both nuclear (ITS1-5.8S-ITS2),and plastid DNA markers and comparison of the results obtained with the data of the traditional taxonomy of this group.

Phylogenetic Analysis of nrITS Dataset
On the nrITS phylogenetic tree, the genus Lotus forms a well-supported clade with several subclades (Figure 2).Among them, a Southern clade, several northern clades (i.e., Lotus Northern clades I, II, III and IV) and a separate branch of Lotus glinoides are observed (Figure 2).Species of the Lotus angustissimus group are clustered within two highly supported clades, Lotus angustissimus group clade 1 and Lotus angustissimus group clade 2, which are not related to each other.Clade 1 is a part of the Lotus Northern clade I, and clade 2 corresponds to the Lotus Northern clade III (Figure 2).
Within clade 1, L. praetermissus and L. angustissimus form well-supported speciesspecific subclades.Lotus macrotrichus is clustered with L. praetermissus taking a basal position in the common L. praetermissus plus L. macrotrichus clade.Lotus palustris, L. castellanus and L. lourdes-santiagoi form another clade, where specimens of the latter two species are mixed in a separate smaller subclade, and specimens of L. palustris specimens occupy a basal position.
Clade 2 includes a mixture of studied specimens of L. parviflorus and L. subbiflorus, as well as three specimens with uncertain taxonomic identification (ANG12, ANG14, and ANG17).In the specimen ANG17, two variants of nrITS were found, differing in position 483 of the alignment (T in clone 1/C in clone 2).
Phylogenetic analysis of the nrITS dataset confirmed that the specimen TH1 belongs to the species Lotus rectus L.    2 and Appendix A for voucher information.

Phylogenetic Analysis of the Plastid DNA Dataset
On the phylogenetic tree constructed by the plastid DNA dataset, the genus Lotus is also well defined and subdivided into two large highly supported clades: the Northern clade and the Southern clade (Figure 3).In addition, the Northern clade on the phylogenetic tree, constructed by plastid data, unites all members of Northern clades I, II, III and IV, identified on the tree constructed by nrITS.Lotus glinoides is included in the Southern clade.
On the phylogenetic tree constructed by the plastid DNA dataset, the genus Lotus is also well defined and subdivided into two large highly supported clades: the Northern clade and the Southern clade (Figure 3).In addition, the Northern clade on the phylogenetic tree, constructed by plastid data, unites all members of Northern clades I, II, III and IV, identified on the tree constructed by nrITS.Lotus glinoides is included in the Southern clade.  2 and Appendix A for voucher information.
Representatives of the Lotus angustissimus group occupy positions in three highly supported subclades within the Northern clade, that is, in Clades A, B and C. The composition of none of these three subclades is identical to that of either clade 1 or clade 2 from the nrITS tree.
Clade A on the plastid DNA phylogenetic tree corresponds to a part of clade 1 on the nrITS tree and includes three well separated species-specific groups consisting of specimens of L. angustissimus, L. praetermissus and L. macrotrichus, respectively.
Clade C on the plastid DNA phylogenetic tree corresponds to a part of clade 2 on the nrITS tree and includes L. parviflorus and three specimens of uncertain taxonomic identification (ANG12, ANG14, and ANG17).
Clade B of the plastid DNA phylogenetic tree partially corresponds to clade 1 and clade 2 of the nrITS tree and includes L. palustris, L. castellanus and L. lourdes-santiagoi together with L. subbiflorus.Within clade B, the species L. palustris, L. castellanus, L. lourdes-santiagoi and L. subbiflorus are in an unresolved position.It is worth noting that the sister position in relation to clade B is occupied by Lotus pedunculatus Cav., which is not a member of the Lotus angustissimus group, but represents a separate group of the Lotus section.Clades A and (B + L. pedunculatus) together form a larger, well-supported clade, while clade C occupies a position away from them.
Phylogenetic analysis of the plastid dataset confirmed the belonging of the TH1 sample to the species Lotus rectus L.

Geographic Patterns Observed in the Lotus angustissimus Group
Both nrITS and studied plastid markers confirm the geographical differentiation within the pair of the closely related species Lotus angustissimus and L. praetermissus.The studied specimens of L. angustissimus occupy the southern part of the common range of these two species, mainly around the Mediterranean and Black Seas and in the southern part of the Caspian Sea, and specimens of L. praetermissus are distributed in the northern part, namely in the center and southeast of the European part of Russia, Ukraine and western Kazakhstan (Figures 2, 3 and 4A).The only studied specimen L. macrotrichus from SW Turkey is clustered together with L. praetermissus in both phylogenetic reconstructions, though geographically it is closer to L. angustissimus (Figures 2, 3 and 4A).   2 and Appendix A for voucher information.See Table 2 and Appendix A for voucher information.
Geographic pattern was also observed within the complex of Lotus castellanus, L. lourdes-santiagoi and L. palustris on the nrITS phylogenetic tree.All specimens of L. palustris from the Eastern Mediterranean are unresolved at the base of the common clade of the complex, whereas all specimens from the Western Mediterranean, including L. castellanus and L. lourdes-santiagoi, formed a highly supported subclade.Two specimens of L. castellanus from Spain and Portugal formerly classified within L. palustris (PAL1 and PAL2), but having an intermediate morphologic pattern between typical L. palustris and L. castellanus, were also members of this western subclade (Figures 2, 3 and 4C).In the phylogenetic tree constructed using plastid markers, no phylogenetic or geographical structure was observed in this complex, since these markers do not allow for resolving the relationships in this group of species (Figure 3).
The distribution areas of the studied specimens of Lotus parviflorus and L. subbiflorus overlap significantly (Figure 4B), which is consistent with their clustering on phylogenetic reconstruction using the nrITS dataset (Figure 2).

Discussion
Here, we present the first molecular phylogenetic study that includes all known upto-date species of the Lotus angustissimus group, analyzed by both nuclear and plastid DNA markers.Despite comparatively small datasets studied for each species, we tried to cover different parts of their ranges, and the results obtained allowed us to make several important conclusions concerning phylogenetic relationships, geography and taxonomy in this group.
The phylogenetic reconstructions obtained in this study confirmed the non-monophyletic nature of the Lotus angustissimus group, which was previously assumed in earlier studies of the genus Lotus [16][17][18].However, all representatives of this group are connected in a complex way by common molecular features.The Lotus angustissimus group represents a good example of the discrepancy between nuclear and plastid phylogeny, which may indicate different trends in the evolution of nuclear and plastid lines and the presence of traces of network evolution.

Lotus angustissimus, L. praetermissus and L. macrotrichus
The results of this study by both nuclear and plastid markers clearly support the separation of L. praetermissus from L. angustissimus.This point of view is shared by many authors of regional floras of the territories of the former USSR, Eastern Europe and Central Asia [19][20][21][22][23][24].The opposite concept was expressed by Heyn [3], some Western European authors [5,25,26], and botanists of the Legume Phylogeny Working Group [27] who did not support the segregation of L. praetermissus from L. angustissimus.Detailed morphometric study [6] did not reveal a clear difference between the two species; however, some important characteristics (e.g., seed surface color) were not included in the analysis due to the absence of ripe seeds in the majority of studied specimens.One of the most prominent morphological features distinguishing typical L. praetermissus from typical L. angustissimus is a specific growth pattern: L. praetermissus is usually a small annual plant with a prominent erect main shoot branched in the upper half, whereas L. angustissimus is a more branched annual or perennial plant with ascending shoots.However, a number of transitional forms are observed between the two extreme morphological variants.Moreover, when the plants of L. praetermissus were grown from seeds in the greenhouse with sufficient water supply, they had a habitus more resembling that of L. angustissimus (Kramina, unpublished data).
The genetic separation of L. praetermissus and L. angustissimus is in good agreement with their geographical distribution, and the range of L. praetermissus is confined to more northern and eastern regions.As a whole, the results obtained in this work can serve as an argument in favor of the allocation of two taxa in the rank of independent species.This contradicts the conclusions made earlier only on morphological grounds [6].Apparently, the problem deserves further study with the involvement of analysis of population variability at both the morphological and molecular levels.
The only studied specimen of the local endemic of Western Anatolia, Lotus macrotrichus, is related to Lotus praetermissus based on both nuclear and plastid DNA markers.These results are more or less consistent with morphometric studies [6], where a specimen of L. macrotrichus was morphologically close to L. praetermissus and Turkish specimens of L. angustissimus in many characteristics, but differed from them by the shape of upper leaflets, larger flowers, styles, and fruits.Sister phylogenetic relationships between L. macrotrichus and L. praetermissus do not contradict the recognition of the former as a separate species, but a more comprehensive study is needed for a balanced decision.

Lotus castellanus, L. lourdes-santiagoi and L. palustris
All three species share a common morphological feature, namely, a keel with a curved keel beak.Their close relationship is confirmed by the ITS nuclear marker.Our study demonstrated that L. palustris is paraphyletic in relation to L. castellanus and L. lourdessantiagoi by the nrITS marker, but plastid data cannot resolve the relationships among these three species.
The molecular phylogenetic results obtained in the present study do not allow support for the isolation of L. lourdes-santiagoi from L. castellanus.These two species can be clearly distinguished from each other, but only by one morphological feature-the presence of pubescence on a standard petal.This character is very interesting because it illustrates the phenomenon of the occurrence of morphological features of a neighboring taxon in this taxon.The pubescent back surface of standard petal is known in Lotus section Pedrosia (Lowe) Christ.(in seven species, incl.Lotus chazaliei H. Boissieu, L. loweanus Webb & Berthel., and others) and section Rhyncholotus (Monod) D.D. Sokoloff (all members), as well as in Cytisopsis ahmedii (Batt.& Pit.) Lassen, Tripodion tetraphyllum (L.) Fourr., and Hammatolobium lotoides Fenzl from the tribe Loteae.Within the species Hammatolobium lotoides, some plants have a pubescent standard petal, whereas the others have a glabrous one [28].A similar variability pattern was observed within four species of Lotus section Pedrosia [29].In the Lotus section Lotus, to which representatives of the L. angustissimus group belong, L. lourdes-santiagoi is the only example of a plant with a pubescent standard petal.morphological features of this species in the mentioned individuals, except for a straight keel beak, which is common for several species of the group.The first reason for such variability may be the hybrid nature of the three specimens with hidden morphological features of one parent species.One of the facts in favor of such a conclusion, which can be made for the sample ANG17, is the polymorphism of nrITS.However, this character does not allow identification of the putative parent species, as it was demonstrated for other cases within the genus Lotus (e.g., [30]).The second reason is incomplete lineage sorting, which is in good agreement with the geographical location of mentioned specimens in glacial refugia of the Mediterranean basin [31] and the southern Caspian region [32].As such, the taxonomic status of the specimens ANG12, ANG14 and ANG17 remains uncertain.We prefer to identify them as Lotus spp. or Lotus hybrids.This problem needs further study using an expanded set of molecular markers, as well as various methods of analysis.

Lotus parviflorus
Heyn [3] considered Lotus parviflorus to be the least problematic species within the Lotus angustissimus group due to a set of clear diagnostic features, such as a long straight keel beak and a short fruit not exceeding the calyx.The results obtained in this work confirmed the isolated position of the clade Lotus parviflorus + L. subbiflorus within the genus Lotus in the nrITS phylogenetic trees, which was found in previous studies on a limited dataset [17,18].In the phylogenetic reconstructions made in this study using the plastid DNA dataset, L. parviflorus forms a separate clade, without L. subbiflorus.The inclusion of three specimens with uncertain taxonomic identification in this clade was discussed above.Thus, there is clear evidence of a genetic relationship between L. parviflorus and only one representative of the Lotus angustissimus group, L. subbiflorus, which is consistent with morphological data [6].We are not inclined to combine these two species into one, since the hybrid nature of L. subbiflorus is shown in the present and previous studies.

Plant Material
The molecular study involved 88 specimens, including 53 specimens of Lotus angustissimus group, 32 specimens of other Lotus species representing all main sections of the genus, and 3 specimens of genera Cytisopsis, Hammatolobium and Tripodion, closely related to Lotus.Samples for molecular studies were taken from herbarium specimens stored in herbaria ANK, B, BM, GAZI, LE, MA, MHA, MW, and NSW.Voucher information and GenBank accession numbers are presented in Table 2 and Appendix A. Geographical distribution of specimens included in molecular analyses is presented on a map (Figure 4) prepared using SimpleMappr online software (https://www.simplemappr.net,accessed on 2 December 2023) [33].All the studied specimens of the Lotus angustissimus group were taxonomically identified by morphological features using descriptions and identification keys from the main literary sources [3,6,9].

DNA Extraction, Amplification and Sequencing
DNA was extracted from herbarium specimens (ca.20 mg of leaf tissue) with a NucleoSpin Plant II kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions or using the CTAB method [34].The nrDNA ITS and plastid DNA regions psbA-trnH intergenic spacer, trnL-trnF intergenic spacer (IGS) and trnL intron, and rps16 intron were selected for the analysis because of their utility in Lotus and high variability [17,35,36].The sequences of the nrITS were amplified with primers NNC-18S10, C26A [37], ITS2 and ITS3 [38].The amplification of the psbA-trnH spacer was conducted using primers trnH2 [39] and psbAF [40].The sequences of the trnL-trnF region of plastid DNA were amplified using standard primers "c", "d", "e" and "f" [41], and the sequences of rps16 intron using primers rpsF, rpsR2 [42], Lot-rps16-F and Lot-rps16-intR [35].PCRs were performed in a 0.02 mL mixture containing 10-20 ng DNA, 3.2 pmol of each primer and MasDDTaqMIX (Dialat LTD, Moscow, Russia) containing 0.2 mM of each dNTP, 1.5 mM MgCl 2 , and 1.5 units of SmarTaqDNA polymerase.Amplification of the nrITS region and all plastid DNA regions was performed under the following conditions: hold 94 PCR products were purified using a Cleanup Mini kit (Evrogen, Moscow, Russia) and then used as a template in sequencing reactions with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1.Sequencing was performed on the 3730 DNA Analyzer (Life Technologies, Carlsbad, CA, USA) in the Syntol company (Moscow, Russia).Forward and reverse strands of all samples were sequenced.For the majority of samples, the polymorphism of ITS within one specimen was detected by direct sequencing (without cloning), by the presence of double peaks on electropherogram.ITS sequences of the specimen ANG17 were cloned in the E. coli vector and then sequenced in the Evrogen Joint Stock Company (Moscow, Russia).
The sequences were aligned using MAFFT version 7.215 [43,44] and then adjusted manually in BioEdit version 7.2.5 [45].The matrices of psbA-trnH spacer, trnL-F and rps16 intron plastid DNA regions were concatenated into a single matrix.Gap-rich and ambiguous positions were excluded from the analyses.The aligned data matrices are presented in Supplementary Materials Datasets S1 and S2.

Phylogenetic Analyses
Phylogenetic analyses were performed separately for the nrITS dataset and a concatenated plastid DNA dataset using both maximum likelihood and Bayesian inference methods.
Maximum likelihood analyses were performed in IQ-tree version 1.6.12[46], and internal branch support was assessed using the ultrafast bootstrap [47] with 10,000 resamplings.The GTR + F + G4 model of nucleotide substitutions for plastid data and the SYM + G4 model for nrITS were selected as the most appropriate based on the Bayesian information criterion in the built-in ModelFinder utility [48].
Bayesian inference was performed using MrBayes v. 3.2.6 [49] considering the optimal model of nucleotide substitutions selected by AICc in PAUP version 4.0a [50] for each marker: SYM + Γ (symmetrical model with substitution rate heterogeneity) for nrITS, and GTR + Γ for plastid data.The Bayesian analysis used four independent runs of 25 million generations and four chains sampling every 1000th generation.Non-convergence assessment and burn-in estimation was carried out in VMCMC ver.1.0.1 [51].The first two million generations were discarded as burn-in, and the remaining trees from both runs were combined in a 50% majority-rule consensus tree.

Conclusions
The results of the present phylogenetic study of the Lotus angustissimus group together with a representative set of Lotus species, using nrITS and a set of three plastid DNA regions, clearly demonstrated a non-monophyletic nature of the study group.The nrITS and plastid dataset tree topologies were incongruent in both Bayesian and ML analyses.
Our data demonstrated geographical differentiation between genetic variants within a pair of species Lotus angustissimus L. and L. praetermissus Kuprian., and the southern variants corresponded to the first and the northern ones to the second of the species.The phylogenetic position of the Anatolian endemic Lotus macrotrichus Boiss.has been determined for the first time, which turned out to be close to L. praetermissus, but separated from the latter by a number of mutations both in nrITS and plastid sequences.The position of Lotus subbiflorus Lag. in phylogenetic trees confirms its hybrid nature and implies that its presumed male parent is L. parviflorus Desf.The present phylogenetic results do not allow us to support the isolation of L. lourdes-santiagoi from L. castellanus, which is consistent with the very weak morphological difference between these taxa.The nrITS data indicate a clear geo-graphical structure within the L. castellanus-L.lourdes-santiagoi-L. palustris complex, where the samples of the Western Mediterranean differ from those of the Eastern Mediterranean.
The present study has revealed several trends in genetic variability within the Lotus angustissimus group.In order to assess the limits of this variability and the degree of genetic isolation of individual species, it is necessary to expand the study both with respect to the sample (including the population level) and with respect to the studied genetic markers.

Figure 2 .
Figure 2. Phylogenetic relationships in Lotus with expanded representation of the L. angustissimus group inferred from Bayesian analysis of the nrITS dataset.Branch lengths are proportional to the number of expected nucleotide substitutions: scale bar corresponds to 0.1 substitutions per site.Numbers above branches are posterior probabilities.Numbers below branches or after slashes are bootstrap support values found in maximum likelihood (ML) analysis of the same dataset (values equal or more than 0.6/60% shown).The colored symbols after the names of the samples indicate belonging to different species or hybrids.See Table 2 and Appendix A for voucher information.Within clade 1, L. praetermissus and L. angustissimus form well-supported species-specific subclades.Lotus macrotrichus is clustered with L. praetermissus taking a basal position in the common L. praetermissus plus L. macrotrichus clade.Lotus palustris, L. castellanus and L. lourdes-santiagoi form another clade, where specimens of the latter two species are mixed

Figure 2 .
Figure 2. Phylogenetic relationships in Lotus with expanded representation of the L. angustissimus group inferred from Bayesian analysis of the nrITS dataset.Branch lengths are proportional to the number of expected nucleotide substitutions: scale bar corresponds to 0.1 substitutions per site.Numbers above branches are posterior probabilities.Numbers below branches or after slashes are bootstrap support values found in maximum likelihood (ML) analysis of the same dataset (values equal or more than 0.6/60% shown).The colored symbols after the names of the samples indicate belonging to different species or hybrids.See Table2and Appendix A for voucher information.

Figure 3 .Figure 3 .
Figure 3. Phylogenetic relationships in Lotus with expanded representation of the L. angustissimus group inferred from Bayesian analysis of the plastid DNA dataset.Branch lengths are proportional Figure 3. Phylogenetic relationships in Lotus with expanded representation of the L. angustissimus group inferred from Bayesian analysis of the plastid DNA dataset.Branch lengths are proportional to the number of expected nucleotide substitution: scale bar corresponds to 0.01 substitutions per site.Numbers above branches are posterior probabilities.Numbers below branches or after slashes are bootstrap support values found in ML analysis of the same dataset (values equal or more than 0.6/60% shown).The colored symbols after the names of the samples indicate belonging to different species or hybrids.See Table2and Appendix A for voucher information.

Supplementary Materials:
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants13010101/s1. Dataset S1: nrITS dataset.Dataset S2: Combined plastid dataset (trnL-F, rps16 and psbA-trnH).Author Contributions: T.E.K. contributed to the field collection of samples, all the laboratory work, including DNA extraction, PCR, sequencing, analyses of the data, figure preparation, and manuscript writing and editing.T.R.H. contributed to all the laboratory work, including DNA extraction, PCR, and sequencing.T.H.S. contributed to phylogenetic analyses of the data and to manuscript writing and editing.All authors have read and agreed to the published version of the manuscript.Funding: The study was supported by the Russian Science Foundation, project 23-24-00052.Data Availability Statement: Data are contained within the article or Supplementary Materials.

Table 1 .
Morphological characteristics of species of the Lotus angustissimus group.

Table 2 .
Taxa, sample code, voucher information and GenBank accession numbers of Lotus angustissimus group specimens used in molecular analyses.Herbarium codes according to Index Herbariorum.New sequences indicated by an asterisk.Accessions numbers without an asterisk were taken from GenBank.

Table 3 .
[6]gnostic morphological characteristics of Lotus castellanus, L. palustris and intermediate specimens.Median and lower-upper quartile values (in brackets) are given for each quantitative character for "pure" species (taken from the previous study[6]).Observed values are given for the specimens PAL1 and PAL2.Character states for each character are indicated as follows: C-like in L. castellanus, P-like in L. palustris, C/P-intermediate, ?-unknown.

Table 4 .
[6]gnostic morphological characteristics of Lotus angustissimus, L. subbiflorus and intermediate specimens.Median and lower-upper quartile values (in brackets) are given for each quantitative character for "pure" species (taken from the previous study[6]).Observed values are given for the specimens ANG12, ANG14 and ANG17.Character states for each character are indicated as follows: A-like in L. angustissimusus, S-like in L. subbiflorus, A/S-intermediate.