**2. Content, preservation, and distribution**

The U.S. National Cotton Germplasm Collection (NCGC) currently has approximately 10,000 accessions covering 45 species of *Gossypium* as described by Fryxell [28]. Representation of species within the collection varies significantly, and to some extent reflects historical oppor‐ tunities for collecting or exchange with other collections (Table 1.) However, there has been a conscious effort to focus on fiber producing species that readily hybridize with the cultivated cottons. Accessions of the collection are officially recognized by Plant Introduction (PI) numbers in the NPGS, which maintains a permanent long-term backup inventory, or base collection, in the NCGRP in Fort Collins. However the working, or active, collection in College Station, TX further subdivides the collection to reflect historical organization and taxonomic classification. This additional step facilitates easy organization and retrieval of seed from the temperature controlled vault. The working collection is under the care of a full-time curator and supporting staff with the ARS-SPARC in College Station, Texas and it is the source of *Gossypium* germplasm distributed to the public.

The collection is subdivided to seven different sub-collections that consist of: 1) variety subcollection, primarily *G. hirsutum* (prefixed by SA-), 2) landrace sub-collection, primarily *G. hirsutum* (prefixed by TX-), 3) *G. barbadense* sub-collection, (prefixed by GB-), 4) Asiatic (Agenome species) sub-collection, (prefixed by either A1-or A2-for *G. herbaceum* or *G. arboreum* respectively), 5) wild species sub-collection (prefixed by a taxon specific genome letter-number combination [9]), 6) genetic marker sub-collection, and 7) a base sub-collection (i.e. NCGRP) of all materials in sub-collections1-6 and new plant introductions [3,10]. Sub-collections 1-5 constitute the "working collection", which is routinely seed propagated and distributed by the USDA-ARS at College Station, Texas. Seed of the working collection is maintained at 4°C and 23% relative humidity [11]. Sub-collection 7 is housed at the NCGRP in Fort Collins, CO under conditions more favorable for long-term storage (-20°C, ~5% seed moisture content [12]). The SA sub-collection is short for Stoneville Accession and represents the obsolete cultivar collection originally started at the Delta Branch Experiment Station in Stoneville, MS. The TX (or TEX) sub-collection was started on the campus of Texas A&M University, College Station, TX, to catalogue *G. hirsutum* landraces and various genetic stocks and is the format continued to the present day, exclusively for *G. hirsutum* photoperiodic landraces or tropically collected material. The GB sub-collection originated from *G. barbadense* accessions curated at Phoenix, AZ, and after consolidation with the main collection, it has continued to expand as new acquisitions have been made.

cotton germplasm resources. Plant Introduction centers were established with the Agricultural Marketing Act of 1946 and a National Seed Storage Laboratory (now the National Center for Genetic Resources and Preservation or NCGRP) was established in 1958 for long term backup storage of germplasm. Federal funds and staff also were committed to create working collections throughout the U.S. to specialize in crops. In 1960, the cotton sub-collections were deposited at NGCRP with their passport data, and in the early 1980's they were consolidated in College Station, TX, USA. Increased staff, greenhouses, seed processing and storage facilities were added. The curators continued to add germplasm from publicly donated cultivars and germplasm lines, explorations and exchanges with other collections and to record descriptors on new and existing germplasm. The International Board for Plant Genetic Resources (now Bioversity International) established a set of cotton descriptors in 1980 to serve as a guide for data collection for the U.S. and other collections [8]. Databases for the National Plant Germ‐ plasm System (NPGS) and the collection are managed online by the Germplasm Resources

The U.S. National Cotton Germplasm Collection (NCGC) currently has approximately 10,000 accessions covering 45 species of *Gossypium* as described by Fryxell [28]. Representation of species within the collection varies significantly, and to some extent reflects historical oppor‐ tunities for collecting or exchange with other collections (Table 1.) However, there has been a conscious effort to focus on fiber producing species that readily hybridize with the cultivated cottons. Accessions of the collection are officially recognized by Plant Introduction (PI) numbers in the NPGS, which maintains a permanent long-term backup inventory, or base collection, in the NCGRP in Fort Collins. However the working, or active, collection in College Station, TX further subdivides the collection to reflect historical organization and taxonomic classification. This additional step facilitates easy organization and retrieval of seed from the temperature controlled vault. The working collection is under the care of a full-time curator and supporting staff with the ARS-SPARC in College Station, Texas and it is the source of

The collection is subdivided to seven different sub-collections that consist of: 1) variety subcollection, primarily *G. hirsutum* (prefixed by SA-), 2) landrace sub-collection, primarily *G. hirsutum* (prefixed by TX-), 3) *G. barbadense* sub-collection, (prefixed by GB-), 4) Asiatic (Agenome species) sub-collection, (prefixed by either A1-or A2-for *G. herbaceum* or *G. arboreum* respectively), 5) wild species sub-collection (prefixed by a taxon specific genome letter-number combination [9]), 6) genetic marker sub-collection, and 7) a base sub-collection (i.e. NCGRP) of all materials in sub-collections1-6 and new plant introductions [3,10]. Sub-collections 1-5 constitute the "working collection", which is routinely seed propagated and distributed by the USDA-ARS at College Station, Texas. Seed of the working collection is maintained at 4°C and 23% relative humidity [11]. Sub-collection 7 is housed at the NCGRP in Fort Collins, CO under conditions more favorable for long-term storage (-20°C, ~5% seed moisture content [12]). The SA sub-collection is short for Stoneville Accession and represents the obsolete cultivar collection originally started at the Delta Branch Experiment Station in Stoneville, MS. The TX

and Information Network (GRIN).

168 World Cotton Germplasm Resources

**2. Content, preservation, and distribution**

*Gossypium* germplasm distributed to the public.



To meet the goal of renewing seed of the collection every ten years or less and to ensure the availability of viable seed for distribution, storage, and backup at the NCGRP, accessions of the collection are grown in the Cotton Winter Nursery (CWN) in Tecoman, Mexico, in a local nursery in College Station, or in greenhouses. The majority of the working collection, including photoperiodic and many perennial accessions, are grown, force self-pollinated, and seed increased at the tropical CWN nursery. A large portion of the TX and GB sub-collections are of tropical origin, uncultivated, and are short-day flowering types that require a tropical nursery or a winter greenhouse to induce flowering and reproduction. Wild diploid species (sub-collection 5 of the collection) and recalcitrant accessions with critically low seed numbers must be seed increased in College Station. These high maintenance accessions typically exhibit characteristics such as a perennial growth habit, exerted stigmas, or prerequisite environmen‐ tal stimuli to induce flowering, to name a few. These accessions are typically grown year round in greenhouses to target seed yields of at least 3000 seeds. Accessions are replenished on a 10 year cycle, as determined by viability studies of cottonseed under storage conditions of the working collection, or as indicated from germination testing on the base collection by the

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**2007 2008 2009 2010 2011 2012**

U.S. 768/59 1,537/90 2,300/101 1,702/78 3,200/83 4,900/128 Int'l. 919/26 546/18 450/12 65/10 1,254/10 1,100/12 Total 1,687/85 2,083/108 2,750/113 1,767/88 4,454/93 6,000/140

**Table 2.** Number of accessions/orders distributed from the National Cotton Germplasm Collection (NCGC) in College

Germplasm users in all countries have equal access to the U.S NCGC, and distributions are limited only by the availability of the germplasm at the time of the request and by proper adherence to customs and phytosanitary laws of the U.S. and the requesting country. The policy of free access has grown out of the belief that germplasm is a world heritage to be freely shared. Seed distribution is provided from the working collection at College Station and shows an upward trend in number of requests and amount of accessions distributed yearly (Table 2). Information regarding materials available can be found online at GRIN-GLOBAL (http://

Seed samples are supplied in amounts of 25 self-pollinated seed per sample, if sufficient seed is available for distribution (accessions with a minimum of 500 seed). Cooperators requiring more than 25 seeds must arrange for their own seed increase and in some cases are asked to return excess seed generated (if they are acquiring rare or difficult to manage accessions and can increase seed under controlled conditions with careful self-pollination in greenhouses). Some seed requests go unfilled due to critically low seed amounts, repeated germination difficulties with the seed, or because users request materials that are unavailable (e.g. germ‐ plasm under patent protection or accessions not present in the collection). Some accessions prove difficult to germinate from original or even increased seed, and require treatments such

Station, TX, to requestors from the U.S. and internationally (int'l) in the past six years.

distribution.grin-global.org/gringlobal/search.aspx).

NCGRP.

1 Southern Plains Agricultural Research Center, USDA-ARS, College Station, TX

2 Crop Genetics Research Unit, USDA-ARS, Stoneville, MS

3 Coastal Plain Soil, Water and Plant Research Center, USDA-ARS, Florence, SC

4 Cropping Systems Research Laboratory, USDA-ARS, Lubbock, TX

5 Texas A&M Agrilife, Lubbock, TX, data compiled from 2005-2013

**Table 1.** *Gossypium* germplasm accession holdings, distribution, seed increase, characterization, digital imaging, and evaluation activities of the U.S. National Cotton Germplasm Collection (NCGC) at College Station, USDA-ARS collaborators, and Texas A&M.

To meet the goal of renewing seed of the collection every ten years or less and to ensure the availability of viable seed for distribution, storage, and backup at the NCGRP, accessions of the collection are grown in the Cotton Winter Nursery (CWN) in Tecoman, Mexico, in a local nursery in College Station, or in greenhouses. The majority of the working collection, including photoperiodic and many perennial accessions, are grown, force self-pollinated, and seed increased at the tropical CWN nursery. A large portion of the TX and GB sub-collections are of tropical origin, uncultivated, and are short-day flowering types that require a tropical nursery or a winter greenhouse to induce flowering and reproduction. Wild diploid species (sub-collection 5 of the collection) and recalcitrant accessions with critically low seed numbers must be seed increased in College Station. These high maintenance accessions typically exhibit characteristics such as a perennial growth habit, exerted stigmas, or prerequisite environmen‐ tal stimuli to induce flowering, to name a few. These accessions are typically grown year round in greenhouses to target seed yields of at least 3000 seeds. Accessions are replenished on a 10 year cycle, as determined by viability studies of cottonseed under storage conditions of the working collection, or as indicated from germination testing on the base collection by the NCGRP.

ARS Texas A&M Agrilife5

Working Collection1 CGRU2 SC3 CSRL4

SSR, Seed Oil/Protein Evaluation

*raimondii* 56 315 3 2 4 - - - 3 - - *schwendimanii* 2 1 1 - - - - - -- - *thurberi* 41 268 20 11 9 - - - 5 - 1 *trilobum* 11 118 4 3 6 - - - - - - - (E) *areysianum* 2 13 1 1 1 - - - 1 - - *incanum* 4 22 1 1 - - - - - - - *somalense* 3 65 1 1 1 - - - 1 - - *stocksii* 4 65 2 2 1 - - - 3 - - - (F) *longicalyx* 4 213 4 4 4 - - - 3 - - - (G) *australe* 11 103 2 2 3 - - - 3 - - *bickii* 5 82 1 1 4 - - - 3 - - *nelsonii* 2 11 2 1 1 - - - - - - -

*exiguum* 1 - 1 - 1 - - - -- - *marchantii* 2 - 2 2 1 - - - -- - *nobile* 2 - 2 2 1 - - - -- - *populifolium* 4 2 1 1 1 - - - -- - *pulchellum* 1 3 1 1 1 - - - -- - *rotundifolium* 1 - 1 - - - - - -- - -

**Table 1.** *Gossypium* germplasm accession holdings, distribution, seed increase, characterization, digital imaging, and evaluation activities of the U.S. National Cotton Germplasm Collection (NCGC) at College Station, USDA-ARS

CLCV Evaluation

Landrace Evaluation

Salt/Drought Evaluation

Characterization, Digital Imaging

Abiotic Evaluation

Biotic Evaluation

Phenotypic Evaluation

*Gossypium* genome/species

(K) *costulatum*

Accessions

170 World Cotton Germplasm Resources

Distribution 1987-2013

Seed Increase 2011-2013

1 Southern Plains Agricultural Research Center, USDA-ARS, College Station, TX

3 Coastal Plain Soil, Water and Plant Research Center, USDA-ARS, Florence, SC

2 Crop Genetics Research Unit, USDA-ARS, Stoneville, MS

collaborators, and Texas A&M.

4 Cropping Systems Research Laboratory, USDA-ARS, Lubbock, TX 5 Texas A&M Agrilife, Lubbock, TX, data compiled from 2005-2013

Characterization, Digital Imaging 2011-2013


**Table 2.** Number of accessions/orders distributed from the National Cotton Germplasm Collection (NCGC) in College Station, TX, to requestors from the U.S. and internationally (int'l) in the past six years.

Germplasm users in all countries have equal access to the U.S NCGC, and distributions are limited only by the availability of the germplasm at the time of the request and by proper adherence to customs and phytosanitary laws of the U.S. and the requesting country. The policy of free access has grown out of the belief that germplasm is a world heritage to be freely shared. Seed distribution is provided from the working collection at College Station and shows an upward trend in number of requests and amount of accessions distributed yearly (Table 2). Information regarding materials available can be found online at GRIN-GLOBAL (http:// distribution.grin-global.org/gringlobal/search.aspx).

Seed samples are supplied in amounts of 25 self-pollinated seed per sample, if sufficient seed is available for distribution (accessions with a minimum of 500 seed). Cooperators requiring more than 25 seeds must arrange for their own seed increase and in some cases are asked to return excess seed generated (if they are acquiring rare or difficult to manage accessions and can increase seed under controlled conditions with careful self-pollination in greenhouses). Some seed requests go unfilled due to critically low seed amounts, repeated germination difficulties with the seed, or because users request materials that are unavailable (e.g. germ‐ plasm under patent protection or accessions not present in the collection). Some accessions prove difficult to germinate from original or even increased seed, and require treatments such as seed scarification or hot water baths to achieve germination. Regular re-inventory of the critical and/or difficult to manage accessions indicate if they need to be re-established from backup seed at the NCGRP, or through a repeated exploration or germplasm exchange with the host country.

last century are not well documented, and published information is limited. A series of collection/exploration trips were made in southern Mexico and Central America in the midtwentieth century by T.R. Richmond and C. W. Manning in 1946, S. G. Stephens in 1947, and J. O. Ware and C. W. Manning in 1948 [21]. From the germplasm collected in these trips, a series of eight 'landraces' was established that generally corresponded to areas of native production or geographic occurrence. N. Lemeshev (USSR) and Q. Obispo (Mexico) in 1977-1978 made several collection-expeditions covering 16 Mexican states, from the peninsula of Baja California to the state of Yucatan. They collected several landraces and seed of seven *Gossypium* species (*G*. *armourianum* Kearney*, G*. *harknessii* Brandegee*, G. trilobum* (DC.) Skovsted*, G*. *aridum* (Rose &Standley) Skovsted*, G*. *lobatum* Gentry, *G*. *laxum* Phillips, and *G*. *gossypioides* (Ulbrich) Standley) [26]*.* P.A. Fryxell from 1968 to 1975 made several collectionexpeditions in the country, providing a larger number of specimens to herbaria (MEXU and XAL) with clear and precise descriptions of habitat and location of collected accessions [27]. He made the most recent taxonomic classification of *Gossypium* species [28,29]. A. E. Percival and J. M. Stewart of the USDA-ARS, and A. Hernandez and F. de Leon of the Instituto Nacional de Investigaciónes Forestales Agricolas y Pecuarias (INIFAP) made several collectionexpeditions throughout the states of the Yucatan Peninsula and in parts of the states of Tamulipas, Veracruz, Tabasco, Oaxaca and Chiapas in 1984 [10]. Also, A. E. Percival (USDA-ARS), J. M. Stewart (Univ. of Arkansas), E.A. Garcia, and L. Peréz (SAGAR-INIFAP Mexico) made additional collection expeditions in the state of Baja California Sur and parts of Sonora

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The most recent collecting efforts in Mexico for the collection were made by M. Ulloa (USDA), J.M. Stewart (Univ. of Arkansas), and several researchers from Mexico's INIFAP. These collecting efforts covered the Pacific west coast of Mexico, from the state of Sonora (North) to the state of Chiapas (South) and included several trips from 2002-2004 [19,26]. As a result of these efforts, a significant number of additional *Gossypium* accessions from various parts of Mexico are now available for evaluation, including several accessions of each diploid species of the D genome [19,26]. The cooperative collecting efforts of 2002-2006 also led to a significant number of additional *Gossypium* accessions of the subgenus *Houzingenia* from various parts of Mexico being placed in a nursery or botanical garden in Iguala Guerrero Mexico, including several accessions of each of the arborescent species for ex *situ* conservation [19,26]. This botanical garden was supported from 2006 through 2009 by additional funding from Cotton Incorporated (Cary, NC, USA) provided by J. M. Stewart to INIFAP. Today, Mexico maintains

this *Gossypium* nursery in Iguala Guerrero (C. Perez-M. *personal communication*).

Since the first collecting trips were made in Mexico, the *in situ* survival of Mexican cotton germplasm has been threatened with increasing human population, modernization of agriculture and urbanization. New roads and population growth continue to increase. At this point, one species (*G. aridum*) of the subsection *Erioxylum* appears not to be threatened, probably because of the great diversity (botanical and geographic) encompassed by this species [26]. However, some of the most recent collected and non-described taxons (e.g., US-72) or ecotypes of the *G. aridum* species [19,26] may be in the process of becoming extinct in the wild. In addition, the D8 *G. trilobum* species is almost extinct or already extinct. The natural habitat

and Sinaloa states in 1990.

#### **3. Collecting and exchange**

Although domestic upland (*G. hirsutum)* cotton dominates world production (around 95%), it is generally accepted that its genetic diversity is very low [13-16]. Various bottlenecks and restrictions to genetic variability have occurred during the breeding history of upland cotton that have led to the low diversity currently seen in commercial cotton [17,18]. However, our germplasm collections have historically served as reservoirs of diversity for the breeding community; providing useful variation for disease and insect resistance, fiber quality im‐ provement, and resistance to environmental stress. Collecting and exchange efforts are necessary to maintain and increase the genetic diversity of our collections [18,19]. In addition to early collecting trips that provided the foundation for the germplasm collection, numerous sponsored plant exploration trips and germplasm exchange efforts have occurred since the mid-twentieth century [10,20,21] and continue to occur. In 1982 the sovereign rights of countries over their germplasm was recognized [22,23] and many countries have become signatories to the International Treaty on Plant Genetic Resources for Food and Agriculture, implemented on 29 June 2004. This treaty states as a goal "the conservation and sustainable use of plant genetic resources for food and agriculture and the fair and equitable sharing of the benefits arising out of their use, in harmony with the Convention on Biological Diversity, for sustainable agriculture and food security". The acquisition of germplasm through collec‐ tion has become the subject of extensive negotiation with host countries, and often includes the topics of benefit sharing and agreements on proprietary rights of host countries to products originating from germplasm. The latter topic has become a major stumbling block to collecting for the U.S. NCGC. In the past, the collection has only acquired germplasm that could be distributed free and clear of proprietary obligations, and the collection remains committed to this policy. Careful bilateral negotiation with individual nations, assuring mutual benefit, has led to limited germplasm acquisitions by the U.S. national collection in the latter half of the twentieth century. Since 1985 twelve acquisition trips have been made by U.S. scientists to collect germplasm. Ten of the trips were used for *in situ* explorations, while two of the trips were conducted to exchange germplasm with India, China, Russia, and Uzbekistan respec‐ tively [24]. Continued collection and germplasm exchange is essential to address gaps in individual collections worldwide and to conserve *ex situ* germplasm threatened by develop‐ ment [25].

The two most recent collecting efforts that resulted in germplasm acquisition by the U.S. collection were to Mexico in 2004-2006 and to Puerto Rico in 2013. Mexico encompasses the species range of several diploid cotton species, and Mexico-Guatemala is the recognized center of origin of *G. hirsutum,* the most widely cultivated species in the world. Mesoamerica is the center of morphological diversity for *G. hirsutum* [26]. Collecting efforts in Mexico during the last century are not well documented, and published information is limited. A series of collection/exploration trips were made in southern Mexico and Central America in the midtwentieth century by T.R. Richmond and C. W. Manning in 1946, S. G. Stephens in 1947, and J. O. Ware and C. W. Manning in 1948 [21]. From the germplasm collected in these trips, a series of eight 'landraces' was established that generally corresponded to areas of native production or geographic occurrence. N. Lemeshev (USSR) and Q. Obispo (Mexico) in 1977-1978 made several collection-expeditions covering 16 Mexican states, from the peninsula of Baja California to the state of Yucatan. They collected several landraces and seed of seven *Gossypium* species (*G*. *armourianum* Kearney*, G*. *harknessii* Brandegee*, G. trilobum* (DC.) Skovsted*, G*. *aridum* (Rose &Standley) Skovsted*, G*. *lobatum* Gentry, *G*. *laxum* Phillips, and *G*. *gossypioides* (Ulbrich) Standley) [26]*.* P.A. Fryxell from 1968 to 1975 made several collectionexpeditions in the country, providing a larger number of specimens to herbaria (MEXU and XAL) with clear and precise descriptions of habitat and location of collected accessions [27]. He made the most recent taxonomic classification of *Gossypium* species [28,29]. A. E. Percival and J. M. Stewart of the USDA-ARS, and A. Hernandez and F. de Leon of the Instituto Nacional de Investigaciónes Forestales Agricolas y Pecuarias (INIFAP) made several collectionexpeditions throughout the states of the Yucatan Peninsula and in parts of the states of Tamulipas, Veracruz, Tabasco, Oaxaca and Chiapas in 1984 [10]. Also, A. E. Percival (USDA-ARS), J. M. Stewart (Univ. of Arkansas), E.A. Garcia, and L. Peréz (SAGAR-INIFAP Mexico) made additional collection expeditions in the state of Baja California Sur and parts of Sonora and Sinaloa states in 1990.

as seed scarification or hot water baths to achieve germination. Regular re-inventory of the critical and/or difficult to manage accessions indicate if they need to be re-established from backup seed at the NCGRP, or through a repeated exploration or germplasm exchange with

Although domestic upland (*G. hirsutum)* cotton dominates world production (around 95%), it is generally accepted that its genetic diversity is very low [13-16]. Various bottlenecks and restrictions to genetic variability have occurred during the breeding history of upland cotton that have led to the low diversity currently seen in commercial cotton [17,18]. However, our germplasm collections have historically served as reservoirs of diversity for the breeding community; providing useful variation for disease and insect resistance, fiber quality im‐ provement, and resistance to environmental stress. Collecting and exchange efforts are necessary to maintain and increase the genetic diversity of our collections [18,19]. In addition to early collecting trips that provided the foundation for the germplasm collection, numerous sponsored plant exploration trips and germplasm exchange efforts have occurred since the mid-twentieth century [10,20,21] and continue to occur. In 1982 the sovereign rights of countries over their germplasm was recognized [22,23] and many countries have become signatories to the International Treaty on Plant Genetic Resources for Food and Agriculture, implemented on 29 June 2004. This treaty states as a goal "the conservation and sustainable use of plant genetic resources for food and agriculture and the fair and equitable sharing of the benefits arising out of their use, in harmony with the Convention on Biological Diversity, for sustainable agriculture and food security". The acquisition of germplasm through collec‐ tion has become the subject of extensive negotiation with host countries, and often includes the topics of benefit sharing and agreements on proprietary rights of host countries to products originating from germplasm. The latter topic has become a major stumbling block to collecting for the U.S. NCGC. In the past, the collection has only acquired germplasm that could be distributed free and clear of proprietary obligations, and the collection remains committed to this policy. Careful bilateral negotiation with individual nations, assuring mutual benefit, has led to limited germplasm acquisitions by the U.S. national collection in the latter half of the twentieth century. Since 1985 twelve acquisition trips have been made by U.S. scientists to collect germplasm. Ten of the trips were used for *in situ* explorations, while two of the trips were conducted to exchange germplasm with India, China, Russia, and Uzbekistan respec‐ tively [24]. Continued collection and germplasm exchange is essential to address gaps in individual collections worldwide and to conserve *ex situ* germplasm threatened by develop‐

The two most recent collecting efforts that resulted in germplasm acquisition by the U.S. collection were to Mexico in 2004-2006 and to Puerto Rico in 2013. Mexico encompasses the species range of several diploid cotton species, and Mexico-Guatemala is the recognized center of origin of *G. hirsutum,* the most widely cultivated species in the world. Mesoamerica is the center of morphological diversity for *G. hirsutum* [26]. Collecting efforts in Mexico during the

the host country.

172 World Cotton Germplasm Resources

ment [25].

**3. Collecting and exchange**

The most recent collecting efforts in Mexico for the collection were made by M. Ulloa (USDA), J.M. Stewart (Univ. of Arkansas), and several researchers from Mexico's INIFAP. These collecting efforts covered the Pacific west coast of Mexico, from the state of Sonora (North) to the state of Chiapas (South) and included several trips from 2002-2004 [19,26]. As a result of these efforts, a significant number of additional *Gossypium* accessions from various parts of Mexico are now available for evaluation, including several accessions of each diploid species of the D genome [19,26]. The cooperative collecting efforts of 2002-2006 also led to a significant number of additional *Gossypium* accessions of the subgenus *Houzingenia* from various parts of Mexico being placed in a nursery or botanical garden in Iguala Guerrero Mexico, including several accessions of each of the arborescent species for ex *situ* conservation [19,26]. This botanical garden was supported from 2006 through 2009 by additional funding from Cotton Incorporated (Cary, NC, USA) provided by J. M. Stewart to INIFAP. Today, Mexico maintains this *Gossypium* nursery in Iguala Guerrero (C. Perez-M. *personal communication*).

Since the first collecting trips were made in Mexico, the *in situ* survival of Mexican cotton germplasm has been threatened with increasing human population, modernization of agriculture and urbanization. New roads and population growth continue to increase. At this point, one species (*G. aridum*) of the subsection *Erioxylum* appears not to be threatened, probably because of the great diversity (botanical and geographic) encompassed by this species [26]. However, some of the most recent collected and non-described taxons (e.g., US-72) or ecotypes of the *G. aridum* species [19,26] may be in the process of becoming extinct in the wild. In addition, the D8 *G. trilobum* species is almost extinct or already extinct. The natural habitat of this species has been replaced by intense and extensive agricultural production of guava (*Psidium* spp.). We speculate that the crop of guava and *G. trilobum* must have very similar environmental requirements, particularly as influenced by altitude (around 4,000 feet). Whereas in the past *G. trilobum* was considered to be a common, widely distributed species, based on surveys (J.M. Stewart and M. Ulloa, 2004 expedition), we are of the opinion that urbanization and agricultural development have very severely eroded its habitat, and that the species is becoming extinct in the wild (Drs. Stewart and Ulloa *personal communication*). Within the eight races described in *G. hirsutum,* much of the original diversity existing *in situ* appears to have been lost. According to information obtained from local sources, eradication of naturally occurring landrace, feral, and dooryard cottons was attempted in areas of southern Mexico in the 1980's in efforts to remove perceived insect reservoirs. Apparently all attempts at commercial cotton production since then have been abandoned. No commercial fields of cotton were encountered during expeditions between 2002 and 2004 in the central and southern part of Mexico [19,26]. Currently, with the exception of the northern cotton produc‐ tion regions of Mexico, the diversity of the *G. hirsutum* is limited to feral plants that occur opportunistically in waste areas and as occasional home garden plants maintained by rural peoples or village residents [26].

plants and to document their locations carefully. A local botanist, Duane Kolterman and the curator of the Puerto Rico Herbarium, Jeanine Velez Gavilan, indicated that two species inhabit PR, *G. hirsutum* and *G. barbadense* with probable species introgression in discovered specimens. Only one clear cut *G. barbadense* was seen and it was a dooryard cotton, which we were not able to access. Most of the observed cotton plants were similar to collection accessions classified as race 'marie galante'[31], which is typical of the Caribbean and often produces good fiber. However these plants were not as strongly arborescent as the classic 'marie galante' accession. In addition these plants had characters, such as tapering boll shapes, which were typical of *G. barbadense*, and therefore warrant further inspection. In the southwest portion of Puerto Rico, in the Cabo Roja region, the cotton plants were more variable. In the salt flats at the southwest tip of Puerto Rico, the dominant cotton plant had shorter, coarser and darker fibers than that typically observed on the remainder of the island, and were more characteristic of accessions classified as race 'yucatanense'[31]. The southwest portion of PR faces the Dominican Republic where a sixth tetraploid species is proposed, and a priority site for further exploration and collection. Dooryard cottons were in low frequency on the island and may be declining as cotton products and substitutes become more available for purchase. One resident extolled the virtues of natural cotton because of lack of chemicals used on them, and she demonstrated how they were propagated via cuttings, which may be indicative of a hard seed coat frequent in wild cottons. The digital data collected allows us to assemble a vivid description of wild cotton in Puerto Rico in what appears to be an ever changing landscape and justifies explora‐

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Phenotypic characterization of the national collection has historically served dual purposes. Until the very recent past (when molecular tools became available), phenotypic descriptors were the primary means of describing the diversity contained in the germplasm collection and rationally classifying that variability. The second role of phenotypic descriptors was to assist breeders and others in identifying germplasm of interest in genetic improvement efforts. However, to be useful, phenotypic descriptors needed to be well defined and universally applied. Throughout the history of the collection, this has been a goal that has been striven for but not completely attained. The convention in the past has been to follow, as much as possible, the guidelines set by Bioversity International (formerly IBPGR) in 1980 [33], revised in 1985 and again in 1995 [34] in creating descriptors. Prior to the universal adoption of computers, the internet, and electronic databases, descriptor data files of the collection's holdings were periodically published, with the last publication occurring in 1987 [10]. Presently, data files of descriptors for accessions of the collection can be found in GRIN-GLOBAL at http://distribu‐ tion.grin-global.org/gringlobal/search.aspx or in the CottonGen database at www.cotton‐ gen.org. Descriptors and descriptor sets for phenotyping the collection have not remained static over time, but have evolved as their use has evolved. In 2009, the Cotton Crop Germplasm Committee [14] summarized the need to expand the collection's databases with morphological, agronomic, molecular, and priority trait evaluations to improve user utility of the collection.

tion to ensure their *ex situ* preservation.

**4. Phenotyping and characterization**

Due to its relative proximity to the U. S. and its status as a U.S. territory, Puerto Rico (PR) was a target of opportunity for collecting efforts in 2013. Puerto Rico was revisited in the most recent germplasm collecting effort by James Frelichowski, the curator of the U.S. collection, Louis Prom, of the USDA-ARS, College Station, TX and by collaborators from the USDA-ARS Tropical Agriculture Research Station (TARS) in Mayaguez, PR. Previous reports on wild cotton in the Caribbean [30,31] and the recent taxonomic identification of a new species in the Dominican Republic [32] justified continued exploration for *Gossypium* in Puerto Rico. The current collection inventory indicates 49 accessions originally collected from Puerto Rico and surrounding islands, but GPS locations and details on habitats are lacking. Judging from the limited passport data, the trend was for wild cotton to occur in areas with drier climates in or near coastal regions. Only four Puerto Rican accessions in the collection were observed to have maturity and productivity ratings similar to cultivar checks in the CWN. The Puerto Rican accessions were more characteristic of landrace cottons, and suggest that cotton in Puerto Rico is likely native or is cotton of tropical origin that has become naturalized.

In the most recent collecting efforts in Puerto Rico, attempts to address gaps in the knowledge of the island's diversity and diversity structure were addressed through tagging of all collection sites and resulting accessions with GPS coordinates and with geographic mapping of native cottons. Seventy-nine cotton plants or populations were photographed and tagged with GPS coordinates to give a detailed map based survey of PR. Numerous other cotton plants or populations were sighted and recorded on a GPS map, but not collected due to time restrictions or safety or access concerns. Southern Puerto Rico was much more populated with cotton than the north, but morphological diversity was seen throughout the island and justified the wide search of the island for wild cotton. Differences in fiber length, quality, seed fuzz, and leaf pubescence were evident during collection, and sometimes individual traits were observed to vary among neighboring plants. This confirms the need to collect from individual plants and to document their locations carefully. A local botanist, Duane Kolterman and the curator of the Puerto Rico Herbarium, Jeanine Velez Gavilan, indicated that two species inhabit PR, *G. hirsutum* and *G. barbadense* with probable species introgression in discovered specimens. Only one clear cut *G. barbadense* was seen and it was a dooryard cotton, which we were not able to access. Most of the observed cotton plants were similar to collection accessions classified as race 'marie galante'[31], which is typical of the Caribbean and often produces good fiber. However these plants were not as strongly arborescent as the classic 'marie galante' accession. In addition these plants had characters, such as tapering boll shapes, which were typical of *G. barbadense*, and therefore warrant further inspection. In the southwest portion of Puerto Rico, in the Cabo Roja region, the cotton plants were more variable. In the salt flats at the southwest tip of Puerto Rico, the dominant cotton plant had shorter, coarser and darker fibers than that typically observed on the remainder of the island, and were more characteristic of accessions classified as race 'yucatanense'[31]. The southwest portion of PR faces the Dominican Republic where a sixth tetraploid species is proposed, and a priority site for further exploration and collection. Dooryard cottons were in low frequency on the island and may be declining as cotton products and substitutes become more available for purchase. One resident extolled the virtues of natural cotton because of lack of chemicals used on them, and she demonstrated how they were propagated via cuttings, which may be indicative of a hard seed coat frequent in wild cottons. The digital data collected allows us to assemble a vivid description of wild cotton in Puerto Rico in what appears to be an ever changing landscape and justifies explora‐ tion to ensure their *ex situ* preservation.
