**3. Collections/explorations and unclassified taxa**

**Figure 5.** Shrubs of *G. turneri* in their natural habitat showing green vegetation after extended drought.

212 World Cotton Germplasm Resources

x 1.8 mm with blackish color and compressed pubescence (Table 1; [12]).

*Gossypium turneri***(D10)** is adapted to the coast of the state of Sonora Mpio. of Guaymas and primarily associated with soils of weathered igneous concentration (Fig. 1). On a long-term basis, *G. turneri* is well adapted to the sea-shore environments and sea level altitude in which it occurs. Based on a recent exploration (J.M. Stewart and M. Ulloa, 2004 expedition), the species has salt and drought resistance mechanisms that allow it to survive extended periods without rain. During this trip, the first *G. turneri* bush encountered was actually in the yard/sea-cliff. Although the species had a very small yield of seed the year when it was encountered because of drought, no evidence was seen of plants that died from the lack of water. Like most other *Gossypium*, in those areas where the species occurs, the plants are quite numerous. Unless unforeseen expansion of the resort industry occurs along the coastal region north of La Manga, the species habitat, in general, probably will not undergo rapid degradation. If resort con‐ struction should occur on the sea cliffs and adjoining valleys, then the species most likely would be lost in the wild. This species can be described as a shrub of around 1 m tall. Figure 5 presents shrubs in their natural habitat still showing green vegetation after extended drought. The species for the most part contains cordate leaves with yellow flowers, filaments and anthers. The seeds are contained in a capsule with three-to five-cells and seeds averaging 3.8

The gene pool of Upland/Acala *G. hirsutum* from the country of Mexico derived one of the primary sources for improvement of most of the Acala and Upland cotton growing in the world today. In addition, another cotton genetic resource of this country is the 11 formally reported D diploid *Gossypium* species and several unclassified taxa [8-11] of the Western Hemisphere. Mexico and its boundaries are the center of diversity of these endemic species. Some of these species and their genomes (US-72, D4, D7, D9, D10, and D11) with arborescent or shrub growth habits express unique flowering and fruiting habits (following defoliation in the dry season) and salt and drought resistance mechanisms that allow them to survive extended periods without rain.

Because of the importance of the gene pool of *G. hirsutum* from Mexico, the collection/ exploration trips of the D diploid species have been difficult to execute and document. Two of the greatest explorers and taxonomists of the *Gossypium* genus, and especially species from the country of Mexico, were Drs. Fryxell and Stewart. P.A. Fryxell made several collectionexpeditions from 1968 to 1975 in the country, providing a larger number of specimens to several Herbariums (Herbarium Nacional de Mexico-MEXU and Herbarium Instituto de Ecologia A.C. Mexico-XAL) with clear and precise descriptions of habitat and location of collected accessions [5]. He also made the most recent taxonomic classification of *Gossypium* species [7]. A. E. Percival, J. M. Stewart (USDA), A. Hernandez and F. de Leon (INIFAP) made several collection-expeditions in 1984 throughout the states of the Yucatan Peninsula and in parts of the states of Tamaulipas, Veracruz, Tabasco, Oaxaca and Chiapas. Also, A. E. Percival (USDA-ARS), J. M. Stewart (Univ. of Arkansas), E.A. Garcia, and L. Peréz (INIFAP, Mexico) made additional collection-expeditions in 1990 in the state of Baja California Sur and parts of the states of Sonora and Sinaloa. As a result of their early efforts, a number of *Gossypium* accessions of the subgenus *Houzingenia* from various parts of Mexico were deposited in the USDA-ARS Cotton Germplasm Collection College Station, TX, USA. Also, during the 1980s, Dr. Lemeshev of the Academy of Science of Russia established a *Gossypium* nursery in Iguala City, state of Guerrero in the country of Mexico. Also, some or all of these species are catalogued in the germplasm collection of the Vavilov Institute in St. Petersburg and in several collections of Former Soviet Union countries (e.g., Uzbekistan) based on several collection-expeditions by the Universidad Autónoma de Guerrero Mexico and the Academy of Science of Russia in the states of Veracruz, Tabasco, Campeche, Yucatán, Chiapas, Guerrero, Oaxaca, Michoacán, Morelos, Colima, Sinaloa, Sonora and Baja California Sur between 1989 and 1993 by F. Talipov, C. Cataláio, F. Salgado and M. Bahena. This nursery was abandoned upon Dr. Lemeshev's return to Russia (Q. Obispo, personal communication; [8]).

Until recently no national resources were dedicated to the preservation of this natural treasure [8-9]. In 2002-2006, the United States Department of Agriculture – Agriculture Research Service (USDA-ARS) and the Mexican Instituto Nacional de Investigaciónes Forestales Agricolas y Pecuarias (INIFAP) sponsored joint *Gossypium* germplasm collection trips by U.S. and Mexican cotton scientists. As a result of these efforts, a significant number of *Gossypium* accessions of the subgenus *Houzingenia* from various parts of Mexico were collected (Table 2). Collected


accessions were placed in a nursery or botanical garden in Iguala, Guerrero, Mexico, including several accessions of each of the arborescent species for ex *situ* conservation. Today, Mexico maintains this *Gossypium* nursery in Iguala, Guerrero (C. Perez-Mendoza. *personal communi‐ cation*). Since the first collection-expedition trips were made, the *in situ* survival of these diploid species has been threatened by increasing human population, modernization of agriculture and urbanization. If *in situ* diversity of the Mexican cottons is severely eroded, then current and additional accessions in all the germplasm collections all over the world and the USDA Cotton Germplasm Collection assume a highly significant role in the preservation of the diversity previously residing in Mexico's dooryard (*G. hirsutum*) and cotton species of the D

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215

As formally reported, *G. aridum* is the most widely distributed wild *Gossypium* species in Mexico [5,7]. However, recent additional collections (http://www.lbk.ars.usda.gov/psgd/ index-cotton.aspx) and several studies [8-11] have reported and suggested non-described taxa and ecotypes that can be considered separate species. Morphological comparisons among specimens of on-site observations indicate extensive differences in leaf size, vestiture of the leaves, morphology in the lysigenous glands on the capsules, and period of flowering. Molecular comparisons have provided useful information in the geographical, taxonomical distribution, and evolutionary history of the New World D genome-species. Based on molec‐ ular data (see additional information in the section below), in addition to previously D4-11-G, US-072 reported new taxon, five new collected accessions [D4-10-O (US-41), D4-2-P (US-05), D4-12- G (US-76), D4-19-C (US-122), and D4-32-N (US-150)] from five different geographical sites (ecotypes) from the states of Oaxaca, Puebla, Guerrero, Colima, and Nayarit may be recognized as new species [8-9]. Subsequent observations on greenhouse plants and a return visit to the sites when the plants are beginning to flower will taxonomically confirm that these populations represent

When traditional taxonomy based on morphology (plant canopy, plant height, leaf and capsule shapes, flowers and petal spots, seed size, etc) do not distinguish two species with intermediate phenotypes, molecular methods provide an alternative solution of resolving these not well defined morphological differences between two species [9,14-15]. Molecular markers such as amplified fragment-length polymorphism (AFLP) [10-11] and microsatellites or simple sequence repeats (SSR) have been used to reveal genetic diversity and to distinguish not well defined differences between species or wild relatives [9]. Molecular marker-gene methods have also been used, for example: internal transcribed spacer (ITS) of ribosomal DNA or ribosomal DNA fragment gene-comparisons [16-18], and fragments of chloroplast DNA [19], or repetitive DNA [20-22]. In addition, a few loci of the *Adh* gene [19,23-25], *FAD*2-1 gene [26], and *Ces A1* gene [27] provided insight into the characterization of the *Gossypium* species. Moreover, the phylogeny of the New World diploid *Gossypium* was analyzed based on three independent single-copy genes (*A1341, AdhC,* and *CesA1b*) [28]. These genes were used in previous studies [22-23], showing a high ratio of phylogenetical informative fragment data.

undescribed taxa (new species) belonging to subsection *Erioxylum.*

**4. Molecular characterization of the D genomes**

genome.

a Letter at the end of the Entry ID indicating the state where the accession was collected: C=colima, G=Guerrero, J=Jalis‐ co, N=Nayarit, M=Michoacan, O=Oaxaca, P = Puebla , and S = Sonora ecotypes. This table was published in the follow‐ ing article: Ulloa M, Abdurakhmonov IY, Perez-M C, Percy R, Stewart McDJ. Genetic diversity and population structure of cotton (*Gossypium* spp.) of the New World assessed by SSR markers. Botany 2013;91 251-259. License agreement was provided by the NRC Research Press – Copyright Clearance Center.

**Table 2.** Summary of the 111 accessions used to investigate genetic diversity and population structure of the New World cottons (*Gossypium* spp.).

accessions were placed in a nursery or botanical garden in Iguala, Guerrero, Mexico, including several accessions of each of the arborescent species for ex *situ* conservation. Today, Mexico maintains this *Gossypium* nursery in Iguala, Guerrero (C. Perez-Mendoza. *personal communi‐ cation*). Since the first collection-expedition trips were made, the *in situ* survival of these diploid species has been threatened by increasing human population, modernization of agriculture and urbanization. If *in situ* diversity of the Mexican cottons is severely eroded, then current and additional accessions in all the germplasm collections all over the world and the USDA Cotton Germplasm Collection assume a highly significant role in the preservation of the diversity previously residing in Mexico's dooryard (*G. hirsutum*) and cotton species of the D genome.

As formally reported, *G. aridum* is the most widely distributed wild *Gossypium* species in Mexico [5,7]. However, recent additional collections (http://www.lbk.ars.usda.gov/psgd/ index-cotton.aspx) and several studies [8-11] have reported and suggested non-described taxa and ecotypes that can be considered separate species. Morphological comparisons among specimens of on-site observations indicate extensive differences in leaf size, vestiture of the leaves, morphology in the lysigenous glands on the capsules, and period of flowering. Molecular comparisons have provided useful information in the geographical, taxonomical distribution, and evolutionary history of the New World D genome-species. Based on molec‐ ular data (see additional information in the section below), in addition to previously D4-11-G, US-072 reported new taxon, five new collected accessions [D4-10-O (US-41), D4-2-P (US-05), D4-12- G (US-76), D4-19-C (US-122), and D4-32-N (US-150)] from five different geographical sites (ecotypes) from the states of Oaxaca, Puebla, Guerrero, Colima, and Nayarit may be recognized as new species [8-9]. Subsequent observations on greenhouse plants and a return visit to the sites when the plants are beginning to flower will taxonomically confirm that these populations represent undescribed taxa (new species) belonging to subsection *Erioxylum.*

## **4. Molecular characterization of the D genomes**

**No. of**

214 World Cotton Germplasm Resources

12 *G. herbaceum* A1

32 *G. aridum* D4

10 *G. lobatum* D7

9 *G. trilobum* D8

5 *G. laxum* D9

World cottons (*Gossypium* spp.).

was provided by the NRC Research Press – Copyright Clearance Center.

a

**Accessions Species Genome Entry ID**

 *G. hirsutum* AD1 TM-1 and Acala Maxxa *G. barbadense* AD2 Pima 3-79 *G. tomentosum* AD3 G-tom *G. mustelinum* AD4 G-must *G. darwinii* AD5 G-darw

11 *G. arboreum* A2 A2-8, A2-41, A2-47, A2-61, A2-72, A2-82, A2-106, A2-141, A2-194, A2-234, and A2-241

7 *G. thurberi* D1 D1, D1-4, D1-23,D1-24, D1-35, D1-37, and D1-35XD8-6 5 *G. armourianum* D2 D2-1, D2-2, D2-q, D2-w, and D2-19XD2-17 5 *G. davidsonii* D3 D3-1, D3-2, D3-23, D3-26, and D3-28

3 *G. raimondii* D5 D5-1, D5-2, and D5-3 2 *G. gossypioides* D6 D6-1-O (US043) and D6-2-O (US046)

1 *G. turneri* D10 D10-1-S (US156)

3 *G. shwendimanii* D11 D11-1-M (US083), D11-2M (US084), and D11-3M (US100)

Letter at the end of the Entry ID indicating the state where the accession was collected: C=colima, G=Guerrero, J=Jalis‐ co, N=Nayarit, M=Michoacan, O=Oaxaca, P = Puebla , and S = Sonora ecotypes. This table was published in the follow‐ ing article: Ulloa M, Abdurakhmonov IY, Perez-M C, Percy R, Stewart McDJ. Genetic diversity and population structure of cotton (*Gossypium* spp.) of the New World assessed by SSR markers. Botany 2013;91 251-259. License agreement

**Table 2.** Summary of the 111 accessions used to investigate genetic diversity and population structure of the New

A1-8-1, A1-8-2, A1-5, A1-9, A1-17, A1-18, A1-19, A1-22, A1-23, A1-40, A1-49, and A1-52

D4-1-P (US004), D4-2-P (US005), D4-3-O (US010), D4-4-O (US011), D4-5-O (US012), D4-6-O (US013), D4-7-O (US15), D4-8-O (US016), D4-9-O (US017), D4-10-O (US041), D4-11-G (US072), D4-12-G (US076), D4-13-G (US078), D4-14- G (US080), D4-15-G (US081), D4-16-C (US117), D4-17-C (US120), D4-18-C (US121), D4-19-C (US122), D4-20-C (US126), D4-21-J (US128), D4-22-J (US130), D4-23-J (US136), D4-24-N (US138), D4-25-C (D4-168a), D4-26-C (D4-168b), D4-27-C (D4-168c), D4-28-N (US147), D4-29-N (US148-a), D4-30- N (US148-b), D4-31-N (US149), and D4-32-N (US150)

D7-1-M (US086), D7-2-M (US101), D7-3-M (US103), D7-4-M (US104), D7-5-M (US105), D7-6-M (US106), D7-7-M (US109), D7-8-M (US110), D7-9-M (US111), and D7-10-M (US112)

D8-1-M (US160), D8-2-M (US162), D8-3-M (US163), D8-A, D8-B, D8-1, D8-6, D8-10, and D8-6XD1-35

D9-1-G (US065), D9-2-G (US066), D9-4-G (US068), D9-5-G (US070), and D9-6- M (US098)

When traditional taxonomy based on morphology (plant canopy, plant height, leaf and capsule shapes, flowers and petal spots, seed size, etc) do not distinguish two species with intermediate phenotypes, molecular methods provide an alternative solution of resolving these not well defined morphological differences between two species [9,14-15]. Molecular markers such as amplified fragment-length polymorphism (AFLP) [10-11] and microsatellites or simple sequence repeats (SSR) have been used to reveal genetic diversity and to distinguish not well defined differences between species or wild relatives [9]. Molecular marker-gene methods have also been used, for example: internal transcribed spacer (ITS) of ribosomal DNA or ribosomal DNA fragment gene-comparisons [16-18], and fragments of chloroplast DNA [19], or repetitive DNA [20-22]. In addition, a few loci of the *Adh* gene [19,23-25], *FAD*2-1 gene [26], and *Ces A1* gene [27] provided insight into the characterization of the *Gossypium* species. Moreover, the phylogeny of the New World diploid *Gossypium* was analyzed based on three independent single-copy genes (*A1341, AdhC,* and *CesA1b*) [28]. These genes were used in previous studies [22-23], showing a high ratio of phylogenetical informative fragment data. Even though phylogenetic relationships with these three single-copy genes among species of the D genome still remain unclear, the molecular data supported the recognition of a new D species (US-72) closely related to *G. laxum* [28]. Similar observations were obtained when molecular diversity and phylogenetic relationships were examined among 33 accessions of arborescent *Gossypium* including 23 of *G. aridum* with Random Amplified Polymorphic DNA (RAPD) and AFLP fragments [11].

The phylogenetic analysis grouped all species into distinct phylogenetic groups consistent with genomic origin (Fig. 6). Based on the Wright's *FST* index using AMOVA analyses [9] of the data sets for all-genomes and the diploid New World D-genomes accessions, the differ‐ entiation among the population groups of the different species was highly significant (*P* ≤ 0.0001). A great deal of total genetic variance was attributed to the difference among and within groups, especially within the *G. aridum* population-groups or ecotypes (Table 2 and Fig. 6) [9]. The analysis clustered the diploids of the New World into six sections with the three bushy types [(*Houzingenia* (D1 and D8), *Integrifolia* (D3-d), and *Caducibracteolata* (D2-1, D2-2, and D10)] and three arborescent types [*Erioxylum* (US-72, D4, D7, D9, and D11), *Selera* (D6), and *Austroamerica‐ na* (D5)]. The classification of the formally reported subgenus and species boundaries are wellunderstood [5,7]. These results are in agreement with other molecular studies [23-25,28]. Also, the statistical analysis of s*tructure* test was used in this study [through measurements of *ad hoc* (Δ*K*) quantity of Evanno statistics] to identify real number of K populations for the germplasm accessions (Table 2). The population structure analysis on this study shed light on the emergence and dispersion of the diploids of the New World and agreed with the hypothesis of a rapid radiation of the American diploid cotton linage that took place somewhere in southwestern Mexico, followed by a differentiation-speciation [9,23-25,28]. This radiation might have occurred before the separation of the Baja California peninsula (7-12 million years ago) from the mainland of the country of Mexico [9,28]. The population structure analyses [9] indicated that Baja California peninsula was colonized from two independent lineages, one from the subsection *Intergrifolia* (Q1, D3-accessions) and the second from the subsection *Caducibracteata* (Q2, D2-accessions) (Fig. 7). These two species (*G. harknessii*-D2-accessions and *G. davidsonii*-D3-accessions) are clearly distinguished by many morphological features: leaves,

The Diploid D Genome Cottons (*Gossypium* spp.) of the New World

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217

The population structure analyses [9] with the geographic distribution and morphology of some of these species also supports the hypothesis that the New World D diploid species may derive from five major lineages (Q1-Q5) that eventually radiated and differentiated about 7-8 million years ago through the country of Mexico. Some species [*G. gossypioides* (D6 genome), *G. laxum* (D9 genome), *G. turneri* (D10 genome), and *G. schwendimanii* (D11 genome)] experienced a more recent differentiation event (Fig 7B). Interspecific gene flow has been recognized as an important evolutionary event in plants. It has also been suggested that improbable interspecific introgression and molecular differentiation may have occurred more often than predicted in angiosperm evolution [9-10,28]. Supra-specific coalescence of some alleles in these species may support the mixed sample-group of the D genome accessions (D4, D6, D9, D10, and D11),

The phylogenetic analysis grouped all species into distinct phylogenetic groups of the New World cottons (Fig. 8), consistent with genomic origin and classified species of the *Houzinge‐ nia* subgenus with the six subsections: subsection *Austroamericana* [*G. raimondii* (D5)]; subsec‐ tion *Caducibracteolata* [*G*. *armourianum* (D2-1), *G*. *harknessii* (D2-2), and *G*. *turneri* (D10)]; subsection *Houzingenia* [*G. thurberi* (D1) and *G. trilobum* (D8)]; subsection *Integrifolia* [*G. davidsonii* (D3-d)]; subsection *Erioxylum* [*G. aridum* (D4)*, G. lobatum* (D7)*, G. laxum* (D9)*,* and *G. schwendimanii* (D11)]; and subsection *Selera* [*G*. *gossypioides* (D6)] [3,4,7]. In addition, several non-described taxa of

flowers, seed capsule, pubescence, etc.

experiencing more recent hybridization events.

In 2013 Ulloa et al [9] reported a study of genetic diversity and population structure of cottons (*Gossypium* spp.) of the New World (Table 2). In this study, the genetic diversity and population structure of 111 cotton accessions of *Gossypium* were assessed with SSR markers with wide genome coverage. The species represented five allotetraploids (AD1 – AD5 genomes), 23 Asiatic diploids of the Old World (A1 and A2 genomes), and 82 diploids of the New World subgenus *Houzingenia* (D1 – D11 genomes) species (Table 2).

**Figure 6.** A) Phylogenetic neighbor-joining dendrogram arbitrarily rooted of 111 *Gossypium* accessions representing five allotetraploids (AD1 – AD5 genomes), 23 Asiatic diploids of the Old World (A1 and A2 genomes), and 82 diploids of the New World subgenus *Houzingenia* (D1 – D11 genomes). Phylogenetic tree developed based upon the proportion of alleles between accessions and B) Unrooted neighbor-joining dendrogram of the 111 *Gossypium* accessions. Branch lengths are shown with bootstrap values. Groups of cotton species and accessions specific to each ecotype are colorcoded for simplicity. This figure was published in the following article: Ulloa M, Abdurakhmonov IY, Perez-M C, Percy R, Stewart McDJ. Genetic diversity and population structure of cotton (*Gossypium* spp.) of the New World assessed by SSR markers. Botany 2013;91 251-259. License agreement was provided by the NRC Research Press – Copyright Clear‐ ance Center.

The phylogenetic analysis grouped all species into distinct phylogenetic groups consistent with genomic origin (Fig. 6). Based on the Wright's *FST* index using AMOVA analyses [9] of the data sets for all-genomes and the diploid New World D-genomes accessions, the differ‐ entiation among the population groups of the different species was highly significant (*P* ≤ 0.0001). A great deal of total genetic variance was attributed to the difference among and within groups, especially within the *G. aridum* population-groups or ecotypes (Table 2 and Fig. 6) [9]. The analysis clustered the diploids of the New World into six sections with the three bushy types [(*Houzingenia* (D1 and D8), *Integrifolia* (D3-d), and *Caducibracteolata* (D2-1, D2-2, and D10)] and three arborescent types [*Erioxylum* (US-72, D4, D7, D9, and D11), *Selera* (D6), and *Austroamerica‐ na* (D5)]. The classification of the formally reported subgenus and species boundaries are wellunderstood [5,7]. These results are in agreement with other molecular studies [23-25,28]. Also, the statistical analysis of s*tructure* test was used in this study [through measurements of *ad hoc* (Δ*K*) quantity of Evanno statistics] to identify real number of K populations for the germplasm accessions (Table 2). The population structure analysis on this study shed light on the emergence and dispersion of the diploids of the New World and agreed with the hypothesis of a rapid radiation of the American diploid cotton linage that took place somewhere in southwestern Mexico, followed by a differentiation-speciation [9,23-25,28]. This radiation might have occurred before the separation of the Baja California peninsula (7-12 million years ago) from the mainland of the country of Mexico [9,28]. The population structure analyses [9] indicated that Baja California peninsula was colonized from two independent lineages, one from the subsection *Intergrifolia* (Q1, D3-accessions) and the second from the subsection *Caducibracteata* (Q2, D2-accessions) (Fig. 7). These two species (*G. harknessii*-D2-accessions and *G. davidsonii*-D3-accessions) are clearly distinguished by many morphological features: leaves, flowers, seed capsule, pubescence, etc.

Even though phylogenetic relationships with these three single-copy genes among species of the D genome still remain unclear, the molecular data supported the recognition of a new D species (US-72) closely related to *G. laxum* [28]. Similar observations were obtained when molecular diversity and phylogenetic relationships were examined among 33 accessions of arborescent *Gossypium* including 23 of *G. aridum* with Random Amplified Polymorphic DNA

In 2013 Ulloa et al [9] reported a study of genetic diversity and population structure of cottons (*Gossypium* spp.) of the New World (Table 2). In this study, the genetic diversity and population structure of 111 cotton accessions of *Gossypium* were assessed with SSR markers with wide genome coverage. The species represented five allotetraploids (AD1 – AD5 genomes), 23 Asiatic diploids of the Old World (A1 and A2 genomes), and 82 diploids of the New World subgenus

**Figure 6.** A) Phylogenetic neighbor-joining dendrogram arbitrarily rooted of 111 *Gossypium* accessions representing five allotetraploids (AD1 – AD5 genomes), 23 Asiatic diploids of the Old World (A1 and A2 genomes), and 82 diploids of the New World subgenus *Houzingenia* (D1 – D11 genomes). Phylogenetic tree developed based upon the proportion of alleles between accessions and B) Unrooted neighbor-joining dendrogram of the 111 *Gossypium* accessions. Branch lengths are shown with bootstrap values. Groups of cotton species and accessions specific to each ecotype are colorcoded for simplicity. This figure was published in the following article: Ulloa M, Abdurakhmonov IY, Perez-M C, Percy R, Stewart McDJ. Genetic diversity and population structure of cotton (*Gossypium* spp.) of the New World assessed by SSR markers. Botany 2013;91 251-259. License agreement was provided by the NRC Research Press – Copyright Clear‐

(RAPD) and AFLP fragments [11].

216 World Cotton Germplasm Resources

ance Center.

*Houzingenia* (D1 – D11 genomes) species (Table 2).

The population structure analyses [9] with the geographic distribution and morphology of some of these species also supports the hypothesis that the New World D diploid species may derive from five major lineages (Q1-Q5) that eventually radiated and differentiated about 7-8 million years ago through the country of Mexico. Some species [*G. gossypioides* (D6 genome), *G. laxum* (D9 genome), *G. turneri* (D10 genome), and *G. schwendimanii* (D11 genome)] experienced a more recent differentiation event (Fig 7B). Interspecific gene flow has been recognized as an important evolutionary event in plants. It has also been suggested that improbable interspecific introgression and molecular differentiation may have occurred more often than predicted in angiosperm evolution [9-10,28]. Supra-specific coalescence of some alleles in these species may support the mixed sample-group of the D genome accessions (D4, D6, D9, D10, and D11), experiencing more recent hybridization events.

The phylogenetic analysis grouped all species into distinct phylogenetic groups of the New World cottons (Fig. 8), consistent with genomic origin and classified species of the *Houzinge‐ nia* subgenus with the six subsections: subsection *Austroamericana* [*G. raimondii* (D5)]; subsec‐ tion *Caducibracteolata* [*G*. *armourianum* (D2-1), *G*. *harknessii* (D2-2), and *G*. *turneri* (D10)]; subsection *Houzingenia* [*G. thurberi* (D1) and *G. trilobum* (D8)]; subsection *Integrifolia* [*G. davidsonii* (D3-d)]; subsection *Erioxylum* [*G. aridum* (D4)*, G. lobatum* (D7)*, G. laxum* (D9)*,* and *G. schwendimanii* (D11)]; and subsection *Selera* [*G*. *gossypioides* (D6)] [3,4,7]. In addition, several non-described taxa of

**Figure 7.** Population structure of cotton (*Gossypium* spp.) accessions: (A) Eighty-two D-genome cotton accessions (D1 – D11) population structure plots at K=5 grouped by Q-matrix, (B) grouped by genomic origins of the D-genome acces‐ sions, and C) 111 *Gossypium* accessions representing five allotetraploids (AD1 – AD5 genomes), 23 Asiatic diploids of the Old World (A1 and A2 genomes), and 82 diploids of the New World subgenus *Houzingenia* (D1 – D11 genomes)-Allgenome cotton accessions population structure plot at K=2, where Q1 (red) represents diploid A-genome accessions (A1 and A2), Q2 (green) represent diploid D-genomes (D1 – D11), and red and green represents the amalgamation of the tetraploid AD genomes (AD1 – AD5). This figure was published in the following article: Ulloa M, Abdurakhmonov IY, Perez-M C, Percy R, Stewart McDJ. Genetic diversity and population structure of cotton (*Gossypium* spp.) of the New World assessed by SSR markers. Botany 2013; 91 251-259. License agreement was provided by the NRC Research Press – Copyright Clearance Center.

than in the same clade with the previously proposed sister, *G. raimondii* [13]. This proposed relationship between these two species was based on phylogentic studies with chloroplast (cpDNA) genes. In Figure 8, *G. gossypioides* has been placed at the basal clade-position of the arborescent subsection *Erioxylum* while *G. raimondii* subsection *Austroamericana* shared a cladeposition with species of the *Caducibracteolata* subsection. The clade-position of these two species may indicate two divergent evolutionary events through introgression or hybridization.

**Figure 8.** A) Phylogenetic neighbor-joining dendrogram using the midpoint rooted of the 82 diploids of the New World subgenus *Houzingenia* (D1 – D11 genomes). Phylogenetic tree developed based upon the proportion of alleles

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between accessions.

The arborescent subsection *Erioxylum* is among the most distinctive in the genus [31]. However, the sectional-levels of *G. aridum*, as formally reported [5-8], still remain unresolved. SSR markers have proved to be a powerful tool in elucidating genetic relationships and population structure of these accessions [9]. A proposed genetic distance (GD) minimum threshold of 0.20 [9] may be useful to define a new taxon, and a clear relationship among cotton species or

the New World diploid *G. aridum* species were found to be distanced from their groups or ecotypes from the states of Nayarit, Guerrero, and Oaxaca (Fig. 8). As mentioned before, *G. gossypioides* has been reported with cryptic repeated genomic recombination during speciation, with conflicting morphological, cytogenetic, and molecular evidence of its phylogenetic affinity to other New World cottons [13]. It has been proposed that *G. gossypioides* might hybridize with an African A-genome and/or extinct taxon based on transfer of repetitive DNA [13]. In the neighbor-joining method, trees are constructed by linking together the two operational taxonomic units or in other words – leaves of the tree, or hypothetical taxonomic units that are the closest mutual "neighbors" [29-30]. The phylogenetic resolution of *G. gossypioides* has been found to be inconsistent because this species has been placed within the New World cotton of the D genome in a basal clade-position of the phylogenetic tree rather

**Figure 8.** A) Phylogenetic neighbor-joining dendrogram using the midpoint rooted of the 82 diploids of the New World subgenus *Houzingenia* (D1 – D11 genomes). Phylogenetic tree developed based upon the proportion of alleles between accessions.

than in the same clade with the previously proposed sister, *G. raimondii* [13]. This proposed relationship between these two species was based on phylogentic studies with chloroplast (cpDNA) genes. In Figure 8, *G. gossypioides* has been placed at the basal clade-position of the arborescent subsection *Erioxylum* while *G. raimondii* subsection *Austroamericana* shared a cladeposition with species of the *Caducibracteolata* subsection. The clade-position of these two species may indicate two divergent evolutionary events through introgression or hybridization.

the New World diploid *G. aridum* species were found to be distanced from their groups or ecotypes from the states of Nayarit, Guerrero, and Oaxaca (Fig. 8). As mentioned before, *G. gossypioides* has been reported with cryptic repeated genomic recombination during speciation, with conflicting morphological, cytogenetic, and molecular evidence of its phylogenetic affinity to other New World cottons [13]. It has been proposed that *G. gossypioides* might hybridize with an African A-genome and/or extinct taxon based on transfer of repetitive DNA [13]. In the neighbor-joining method, trees are constructed by linking together the two operational taxonomic units or in other words – leaves of the tree, or hypothetical taxonomic units that are the closest mutual "neighbors" [29-30]. The phylogenetic resolution of *G. gossypioides* has been found to be inconsistent because this species has been placed within the New World cotton of the D genome in a basal clade-position of the phylogenetic tree rather

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218 World Cotton Germplasm Resources

**Figure 7.** Population structure of cotton (*Gossypium* spp.) accessions: (A) Eighty-two D-genome cotton accessions (D1 – D11) population structure plots at K=5 grouped by Q-matrix, (B) grouped by genomic origins of the D-genome acces‐ sions, and C) 111 *Gossypium* accessions representing five allotetraploids (AD1 – AD5 genomes), 23 Asiatic diploids of the Old World (A1 and A2 genomes), and 82 diploids of the New World subgenus *Houzingenia* (D1 – D11 genomes)-Allgenome cotton accessions population structure plot at K=2, where Q1 (red) represents diploid A-genome accessions (A1 and A2), Q2 (green) represent diploid D-genomes (D1 – D11), and red and green represents the amalgamation of the tetraploid AD genomes (AD1 – AD5). This figure was published in the following article: Ulloa M, Abdurakhmonov IY, Perez-M C, Percy R, Stewart McDJ. Genetic diversity and population structure of cotton (*Gossypium* spp.) of the New World assessed by SSR markers. Botany 2013; 91 251-259. License agreement was provided by the NRC Research

> The arborescent subsection *Erioxylum* is among the most distinctive in the genus [31]. However, the sectional-levels of *G. aridum*, as formally reported [5-8], still remain unresolved. SSR markers have proved to be a powerful tool in elucidating genetic relationships and population structure of these accessions [9]. A proposed genetic distance (GD) minimum threshold of 0.20 [9] may be useful to define a new taxon, and a clear relationship among cotton species or

genetically distant geographical accession-ecotypes of *G. aridum.* In addition to US-72 (newly identified taxon) [10,27], five newly collected accessions [D4-10-O (US-41), D4-2-P (US-05), D4-12-G (US-76), D4-19-C (US-122), and D4-32-N (US-150)] from five different ecotypes and states from the country of Mexico were proposed by Ulloa et al [9] to be recognized as new species based on GD. These collected accessions had the larger GD when compared with any other recognized *Gossypium* species of the D genome, GD > 0.28 and GD ≤ 0.41 [9]. Based on the most recent explorations/collections in the country of Mexico [8-9], the existing taxonomic classification of *Gossypium* of the D4 diploid species made by Fryxell [31] and Fryxell et al [7] needs to be revised.

species. The elite public and private cultivars [*G. hirsutum* (Upland) and *G. barbadense* (Pima)] of these species contain a number of traits that originated in the primary germ‐ plasm pool e.g., the blight resistance genes [35], the nectariless trait from *G. tomentosum* [41], root-knot nematode resistance from landraces [42-43], resistance to Fusarium (*Fusarium oxysporum* f.sp. *vasinfectum* Atk. Sny & Hans) and Verticillium (*Verticillium dahlia* Kleb) wilt

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The diploid species of the A and D genome belong to the secondary germplasm pool and have contributed to improving Upland and Pima cultivars [4]. Bacterial blight resistance genes from species such as *G. arboreum*, *G. herbaceum* and *G. anomalum* have been introgressed into Upland cultivars [35]. Cytoplasm and restorer factors from *G. harknessii* [45] and *G. trilobum* [46] conditioning cytoplasmic male sterility, and D2 smoothness [47] have also been introgressed into Upland cultivars using these diploid species. Moreover, improvement of fiber quality characteristics or properties has been done via the triple hybrid (*G. hirsutum* x *G. arboreum* x *G. thurberi*) [48]. Introgression of high fiber strength and improvement of fiber quality param‐ eters were obtained using progeny from these hybrid combinations. In addition, similar triple hybrid combinations that include *G. thurberi* [49] and *G. aridum* [50] have provided progeny that have been used to develop resistant germplasm and cultivars to root-knot nematode (*rkn*, *Meloidogyne incognita* Kofoid and White) and reniform nematode (Renari, *Rotylenchulus reniformis* Linford and Oliveira). Resistance to several pests and diseases has been found in diploid cottons. However, in nature, the hybridization of diploid species with allotetraploid (Upland or Pima) species produces sterile hybrids because uneven genome or chromosome basic number and pairing during meiosis. One of the satisfactory mutagenic agents used by the breeders to induce doubling of chromosomes and balance chromosome paring on hybrids is colchicine. The difficulties of obtaining agronomically suitable introgressed progeny are high through this type of interspecific hybridization. The most successful method of intro‐

Even though the *Gossypium* species of the D genome are not well known and utilized in cotton improvement and breeding, their significance as great reservoirs of important genes is starting to be noticed and documented. In a comparison quantitative trait loci (QTL) review-study [4], the Dt subgenome exhibits from 32% [4,51] to 57% [52] of QTLs on different chromosomes with QTL effects on different important traits for cotton improvements. These QTLs were located on different chromosomes of the Dt subgenome. And even though the species of the D genome does not produce spinnable fibers, the Dt subgenome of the tetraploid cotton was found to possess QTLs positively affecting fiber quality and morphological traits [53-55] and therefore harboring greater allelic diversity among tetraploid forms. Recently, based on the concept that some diploid species are tolerant to stress and may harbor important genes, a large number of genes were obtained from leaf and root tissues of the diploid *G. aridum* species. Plants of this species were subjected to various salt stresses to examine gene expression and to under‐ stand the salt tolerance mechanisms in *Gossypium* [56]. Most of the salt-regulated transcripts were found to be homologous to genes that are known to be associated with salt tolerance e.g., ethylene-responsive transcript factor, aquaporin PIP1, protein kinases (CBL-interacting and mitogen-activated) [56]. New transcriptome data from these plant tissue-species when

from landraces of *G. hirsutum* and from *G. darwinii* [44].

gression has been via hexaploid bridging.
